Gift  of 


D.W.  Bennett,  M.D, 


eo 


Ube  flfeebfcal  Epitome  Series, 

MICROSCOPY  AND  BACTERIOLOGY. 

A  MANUAL  FOR  STUDENTS  AND  PRACTITIONERS, 


BY 

P.  E.^.RCHINARD,   A.M.,  M.  D., 

Demonstrator  of  Microscopy  and  Bacteriology,  Tulane  University  of 
Louisiana,  Medical  Department. 

SERIES   EDITED  BY 
V.  C.  PEDERSEN,  A.M.,  M.  D., 

Instructor  in  Surgery  and  Assistant  Anaesthetist  at  the  New  York  Polyclinic  Medical  School 

and  Hospital;  Deputy  Genito- Urinary  Surgeon  to  the  Out- Patient  Department  of  the 

New    York  Hospital;   Physician-in- Charge,  St.    Chrysostom's   Dispensary; 

Anaesthetist  to  the  Roosevelt  Hospital  (First  Surgical  Division). 

ILLUSTRATED  WITH  SEVENTY-FOUR  ENGRAVINGS. 


LEA  BROTHERS  &  CO., 

PHILADELPHIA    AND    NEW    YORK. 


Entered  according  to  Act  of  Congress,  in  the  year  1903,  by 

LEA  BROTHERS  &  CO., 
In  the  Office  of  the  Librarian  of  Congress.    All  rights  reserved. 


ELECTROTYPED  BY 
3TT  &  THOMSON,  PHILAOA, 


PRESS    OF 
WM.   J.   DORNAN,    PHILADA. 


AUTHOR'S  PREFACE. 


THE  scope  of  an  Epitome  of  Bacteriology  and  Microscopy 
obviously  affords  little  or  no  opportunity  for  original  work, 
nor  indeed  would  it  be  desirable  to  do  more  than  represent 
the  actual  status  of  these  cognate  sciences.  Standard  text- 
books have  accordingly  been  consulted  freely.  The  merit 
of  an  Epitome  consists  in  affording  a  concise  and  clear 
presentation  of  essentials,  command  of  which  enables  the 
student  to  build  a  sound  superstructure  of  knowledge.  The 
practitioner  may  also  use  such  a  volume  to  post  himself 
on  the  main  facts  of  Bacteriology  and  Microscopy,  and  the 
technique.  The  list  of  questions  appended  to  each  chapter 
will  be  useful  to  students  in  quizzing  and  reviewing. 

P.  E.  A. 

NEW  ORLEANS,  1903. 

3 


EDITOR'S  PREFACE. 


IN  arranging  for  the  editorship  of  The  Medical  Epitome 
Series  the  publishers  established  a  few  simple  conditions, 
namely,  that  the  Series  as  a  whole  should  embrace  the  entire 
realm  of  medicine ;  that  the  individual  volumes  should  au- 
thoritatively cover  their  respective  subjects  in  all  essentials ; 
and  that  the  maximum  amount  of  information,  in  letter- 
press and  engravings,  should  be  given  for  a  minimum  price. 
It  was  the  belief  of  publishers  and  editor  alike  that  brief 
works  of  high  character  would  render  valuable  service  not 
only  to  students,  but  also  to  practitioners  who  might  wish 
to  refresh  or  supplement  their  knowledge  to  date. 

To  the  authors  the  editor  extends  his  heartiest  thanks  for 
their  excellent  work.  They  have  fully  justified  his  choice 
in  inviting  them  to  undertake  a  kind  of  literary  task  which 
is  always  difficult — namely,  the  combination  of  brevity,  clear- 
ness, and  comprehensiveness.  The  authors  have  shown  a 
consistent  interest  in  the  work  and  an  earnest  endeavor  to 
cooperate  with  the  editor  throughout  the  undertaking.  Co- 
operation of  this  kind  ought  to  result  in  useful  books,  in 
brief  manuals  as  contradistinguished  from  mere  compends. 
The  editor  desires  at  this  opportunity  to  express  his  appre- 


6  EDITOR'S  PREFACE. 

elation  of  their  helpfulness  in  the  matter  of  producing  the 
proper  character  of  work. 

In  order  to  render  the  volumes  suitable  for  quizzing,  and 
yet  preserve  the  continuity  of  the  text  unbroken  by  the 
interpolation  of  questions  throughout  the  subject-matter, 
which  has  heretofore  been  the  design  in  books  of  this  type, 
all  questions  have  been  placed  at  the  end  of  each  chapter. 
This  new  arrangement,  it  is  hoped,  will  be  convenient  alike 
to  students  and  practitioners. 

VICTOR  C.  PEDERSEN. 
NEW  YORK,  1903. 


CONTENTS. 


INTRODUCTION. 

PAGES 

The  Refraction  of  Light  and  the  Microscope 17-27 

THE  REFRACTION  OF  LIGHT  :  The  Two  Fundamental  Laws  of 

Kefraction ;  The  Principles  of  Refraction  by  Lenses    .    .    .    17-19 
THE  MICEOSCOPE:   The  Simple   Microscope;   The  Compound 
Microscope;  The   Lenses  and   Lens-systems  of  the  Micro- 
scope; The  Care  of  the  Microscope   .    .    .    . 19-27 

CHAPTEE  I. 

The  Fundamental  Principles 27-41 

THE  HISTORY  OF  BACTERIOLOGY 27-28 

THE  CLASSIFICATION  OF  COHN  FOR  BACTERIA ,  28 

THE  DEFINITION  OF  BACTERIA .  28-29 

THE   MORPHOLOGICAL   CLASSIFICATION  OF  BACTERIA:    The 

Coccus ;  The  Bacillus ;  The  Spirillum 29-31 

THE  SIZE  OF  BACTERIA 31 

THE  REPRODUCTION  OF  BACTERIA:  Fission;  Sporulation    .    .  32-34 

THE  MOTILITY  OF  BACTERIA 35 

THE  RELATION  OF  OXYGEN  TO  BACTERIAL  LIFE 36 

THE  RELATION  OF  DEAD  AND  LIVING  ORGANIC  MATTER  TO 

BACTERIA 36 

THE  ESSENTIAL  CONDITIONS  OF  BACTERIAL  GROWTH  :  Heat ; 
Moisture ;  Decomposable  Organic  Material ;  Special  Chemi- 
cal Reaction  of  the  Culture-medium .  36-38 

THE  INERT  AND  INHIBITIVE  CONDITIONS  OF  BACTERIAL  LIFE  38 

THE  VITAL  MANIFESTATIONS  OR  FUNCTIONS  OF  BACTERIA  .  38-41 


8  CONTENTS. 

CHAPTER  II. 

PAGES 

The  Examination  and  the  Staining  of  Bacteria  ......   41-54 

THE  EXAMINATION  OF  BACTERIA  :  The  Hanging-Drop  Prepa- 
ration    41-42 

THE  STAINING  OF  BACTERIA:  The  General  Mode  of  Proced- 
ure ;  The  Most  Commonly  Used  Stains ;  The  Application  of 
the  Dyes ;  The  Special  Methods  of  Staining ;  The  Staining 
of  Capsules;  The  Staining  of  Spores;  The  Staining  of 
Flagella ;  The  Staining  of  Bacteria  in  Tissues 42-54 

CHAPTEE  III. 

The  Process,  Media,  and  Utensils  of  the  Cultivation  of 

Bacteria 55-68 

THE  PROCESS  OF  THE  CULTIVATION  OF  BACTERIA 55 

THE  MEDIA  OF  THE  CULTIVATION  OF  BACTERIA:  The  Most 
Commonly  Used  Liquid  Culture-Media;  The  Most  Com- 
monly Used  Solid  Culture-Media;  The  Most  Commonly 

Used  Special  Culture-Media 55-62 

THE  UTENSILS  OF  THE  CULTIVATION  OF  BACTERIA    ....    62-68 

CHAPTER  IV. 

The  Inoculation  of  Culture-Media  with  Bacteria   .....  68-75 

THE  METHOD  OF  INOCULATING  FLUID  MEDIA 68 

THE  METHODS  OF  INOCULATING  SOLID  MEDIA 68-72 

THE  CULTIVATION  OF  ANAEROBIC  BACTERIA:  The  Incubator 

and  the  Thermostat 72-75 

CHAPTER  V. 

Sterilization,  Disinfection,  and  Antisepsis                    .   .  .  76-84 

THE  METHODS  OF  STERILIZATION 81 

THE  METHODS  OF  DISINFECTION 81-83 

THE  METHODS  OF  ANTISEPSIS:  The  Common  Disinfectants    .  83-84 

CHAPTER  VI. 

The  Inoculation  of  Animals  and  their  Study                     .    85-91 
THE  INOCULATION  OF  ANIMALS  :  The  Various  Methods  of  In- 
oculation of  Animals  .    .        85-88 

THE  OBSERVATION  OF  THE  INOCULATED  ANIMAL  :  The  Roux- 

Nocard  Method  of  Culture  and  Observation  .  .    89-91 


CONTENTS.  9 

CHAPTER  VII. 

PAGES 

Infection  and  Immunity 91_99 

INFECTION:   The  Theories  of  Infection;   The   Avenues  and 

Factors  of  Infection 91-94 

IMMUNITY  AND  ITS  VARIETIES  :  The  Methods  of  Producing 
Immunity;  The  Antitoxic  and  Antimicrobic  Blood- 
Serums;  The  Thories  of  Immunity 94-99 

CHAPTER  VHI. 

The  Pathogenic  Bacteria 99-107 

THE  PYOGENIC  MICROCOCCI  AND  ALLIED  BACILLI  ....  99-100 
THE  INDIVIDUAL  FEATURES  OF  THE  PYOGENIC  BACTERIA  : 
Staphylococcus  Pyogenes  Aureus;  Staphylococcus  Pyo- 
genes  Albus ;  Staphylococcus  Citreus;  Streptococcus 
Pyogenes ;  The  Micrococcus  Cereus  Albus ;  The  Micrococ- 
cus  Cereus  Flavus ;  The  Micrococcus  Pyogenes  Tenuis  ; 

Micrococcus  Tetragenus 100-104 

GONORRHCEA:  Micrococcus  Gonorrhoea  (Gonococcus) ;  Bacil- 
lus Pyocyaneus;  Bacillus  Pyogenes  Fcetidus;  Pneumo- 
coccus  or  Pneumobacillus ;  Bacillus  Coli  Communis , 
Bacillus  Typhosus;  Bacillus  Tuberculosis 104-107 

CHAPTER  IX. 

The  Other  Pathogenic  Micrococci  and  Allied  Bacilli— 
Micrococcus  Pneumonia,  Epidemic  Cerebrospinal  Men- 
ingitis, and  Malta  Fever 108-115 

PNEUMONIA:  Micrococcus  Pneumonise  Crouposae  (Diplococ- 
cus  Pneumonias ;  Micrococcus  Pasteuri ;  Micrococcus  of 
Sputum  Septicaemia) ;  Pneumococcus  of  Friedlaender 

(Bacillus  Pneumonise  of  Fluegge) 108-112 

EPIDEMIC  CEREBROSPINAL  MENINGITIS:  Diplococcus  Intra- 

cellularis  Meningitidis 112-113 

MALTA  OR  MEDITERRANEAN  FEVER:  Micrococcus  Melitensis    113-115 

CHAPTER  X. 

Tuberculosis 115-120 

BACILLUS  TUBERCULOSIS  .  115-120 


10  CONTENTS. 

CHAPTEE  XL 

PAGES 

Leprosy  and  Syphilis   ....                      120-123 

LEPROSY:  Bacillus  Leprae 120-122 

SYPHILIS  :  Bacillus  of  Syphilis ;  Streptococcus  of  Syphilis    .  122-123 

CHAPTER  XII. 

Glanders  (Farcy) 12^128 

BACILLUS  MALLEI 124-128 

CHAPTEE  XIIL 

Anthrax 128-133 

BACILLUS  ANTHRACIS 128-133 

CHAPTEE  XIV. 

Diphtheria  and  Pseudodiphtheria 133-145 

DIPHTHERIA:  Bacillus  Diphtherias 133-140 

PSEUDODIPHTHERIA:  Bacillus  Pseudodiphtheriae ;  The  Anti- 
toxin Treatment  of  Diphtheria 140-145 

CHAPTEE  XV. 

Tetanus,  Malignant  (Edema,  and  Symptomatic  Anthrax  145-156 

MALIGNANT  (EDEMA:  The  Bacillus  of  Malignant  (Edema  .  152-153 

SYMPTOMATIC  ANTHRAX:  Bacillus  Anthracis  Symptomatici  .  153-156 

CHAPTEE  XVI. 

Typhoid  Fever 156-165 

BACILLUS  TYPHOSUS 156-159 

DIFFERENTIATION  OF  BACILLUS  TYPHOSUS  FROM  ALLIED 

GROUPS 159-162 

THE  BLOOD-SERUM  DIAGNOSIS  OF  TYPHOID  FEVER     .    .    .  162-164 

VACCINATION  AGAINST  TYPHOID  FEVER 164-165 

CHAPTEE  XVII. 

Bacillus  Coli  Communis 165-168 

CHAPTEE  XVHL 

Asiatic  Cholera 168-174 

SPIRILLUM  CHOLERA  ASIATICS  (COMMA  BACILLUS)  .   .    .  168-174 


CONTENTS,  11 
CHAPTER  XIX. 

PAGES 

Influenza 174-176 

BACILLUS  OF  INFLUENZA 174-176 

CHAPTER  XX. 

Bubonic  Plague 176-179 

BACILLUS  PESTIS 176-179 

CHAPTER  XXI. 

Relapsing  Fever 179-180 

SPIRILLUM  OBERMEIERI 179-180 

CHAPTER  XXII. 

Dysentry,  Hog  Cholera,  and  Chicken  Cholera 180-185 

DYSENTRY  :  Bacillus  Dysentericae 180-182 

HOG  CHOLERA:  Bacillus  sui  Pestifer 182-183 

CHICKEN  CHOLERA  :  Bacillus  Cholerse  Gallinarum 183-185 

CHAPTER  XXIII. 

The  Pathogenic  Micro-organisms  other  than  Bacteria  .   .  185-196 

ACTINOMYCOSIS,    MALARIA,    AND   AMCEBIC    COLITIS :     StreptO- 

thrix 185-186 

ACTINOMYCOSIS:  Streptothrix  Actinomyces  (Ray  Fungus); 

Other  Pathogenic  Streptothrices  .  , 186-188 

MALARIA:  Plasmodium  Malarise 189-193 

AMCEBIC  COLITIS  :  Amoeba  Coli 194-196 

CHAPTER  XXIV. 

Bacteriological  Examinations  of  Water,  Air,  and  Soil   .  196-204 

THE  BACTERIOLOGICAL  INVESTIGATION  OF  WATER  ....  196-202 

BACTERIOLOGICAL  EXAMINATION  OF  THE  AIR 202-203 

THE  BACTERIOLOGICAL  EXAMINATION  OF  THE  SOIL    .   .   .  203-204 


MICROSCOPY  AND  BACTERIOLOGY. 


INTRODUCTION. 
THE  REFRACTION   OF    LIGHT  AND    THE   MICROSCOPE. 

THE  REFRACTION  OF  LIGHT. 

Definition. — Refraction  is  the  property  possessed  by  trans- 
parent media  of  altering  the  rays  of  light  which  pass  through 
them.  It  is  to  this  property  possessed  by  lenses,  the  trans- 
parent media  of  microscopes,  that  these  instruments  owe 
their  magnifying  power. 

The  Two  Fundamental  Laws  of  Refraction. 

I.  When   a   ray  of  light  passes  from   a   denser  to  a  rarer 
medium,  it  is  refracted  away  from  a  line  drawn  perpendicu- 
larly to  the  plane  which   divides  the  media ;  and  vice  versa, 
when  the  light  passes  from  a  rarer  to  a  denser  medium,  it  is 
refracted  toward  that  perpendicular. 

II.  The  sines  of  the  angles  of  incidence  and  refraction — that 
is,  of  the  angles  which  the  ray  makes  with  the  perpendicular 
before  and  after  its  refraction — bear  to  one  another  a  constant 
ratio  for  each   substance,  which  is    known   as  its  index  of 
refraction. 

The  Principles  of  Refraction  by  Lenses. 

Microscope  lenses  are  chiefly  convex ;  those  of  other  forms 
are  used  to  make  certain  modifications  in  the  rays  passing 
through  the  convex  lens,  and  so  render  their  performance 
more  exact. 

2— M.  B.  17 


1 8  1NTR  OD  UCTlONi 

Focus  of  a  Lens.— Foi1  a  lens  to  give  a  perfect  image  of  an 
object,  all  the  rays  of  light  coming  from  that  object  and  pass- 
ing through  the  lens  must  meet  at  the  same  point  on  the 
other  side  of  the  lens.  This  point  is  known  as  the  focal  point 
or  focus  of  the  lens. 

The  Spherical  Aberration  of  Lenses. 

Definition. — It  is  difficult,  however,  so  to  construct  a  con- 
vex lens  that  all  the  rays  of  light  that  pass  through  it  shall 
come  to  the  same  focus.  As  a  rule,  the  rays  which  traverse 
its  peripheral  or  marginal  portion  come  to  a  shorter  focus 
than  those  which  pass  through  its  more  central  portion. 

Correction. — Distortion  of  the  image  is  thus  caused,  and  is 
known  as  spherical  aberration.  Theoretically,  spherical  aber- 
ration might  be  corrected  by  making  the  curvature  of  the 
periphery  of  the  lens  less  than  that  of  its  more  central  por- 
tion ;  but  the  difficulties  in  the  mechanical  construction  of 
such  a  lens  would  be  very  great,  and  opticians  have  found  it 
more  practical  to  correct  this  defect  by  coupling  with  a  con- 
vex lens  a  concave  one  of  less  curvature,  but  which  is  subject 
to  exactly  the  opposite  error  of  refraction. 

Doublets. — These  combinations  of  convex  and  concave 
lenses,  or  doublets,  act  as  a  single  convex  lens. 

Triplets. — Sometimes  two  convex  and  one  concave  lens  are 
used  in  combination,  and  are  called  triplets. 

The  Chromatic  Aberration  of  Lenses. 

Definition. — When  light  traverses  a  convex  lens,  the  differ- 
ent colors  which  compose  it  do  not  all  come  to  the  same  focus 
— that  is,  the  colors  are  of  unequal  refrangibility ;  and  the 
image  then  is  seen  chiefly  in  that  color  which  chances  to  be 
in  focus.  This  color-distortion  is  especially  noticeable  with 
the  marginal  rays,  and  is  known  as  chromatic  aberration. 

Correction. — Though  exclusion  of  the  marginal  rays  can, 
as  with  spherical  aberration,  partly  correct  this  defect,  yet 
this  is  not  sufficient,  and  chromatic  aberration  is  remedied 
best  by  constructing  the  convex  lenses  of  the  combination 


THE  MICROSCOPE.  19 

mentioned  above  of  crown  glass,  and  the  concave  lenses  of 
flint  glass,  as  those  two  kinds  of  glass  have  opposite  proper- 
ties with  regard  to  refrangibility. 


THE  MICROSCOPE. 

Microscopes  are  of  two  kinds :  simple  and  compound : 

The  Simple  Microscope. 

The  ordinary  hand  magnifying-glass  and  the  dissecting 
microscope  are  examples  of  simple  microscopes. 

Their  magnifying  power  depends  upon  one  lens  or  several 
lenses  acting  as  one  double  convex  lens.  To  obtain  a  clear, 
enlarged  image  of  the  object,  the  latter  must  be  in  its  princi- 
pal focus,  and  the  shorter  the  focus  of  the  lens  the  greater  its 
magnifying  power.  The  focal  length,  or  focus,  of  the  lens 
depends  on  the  degree  of  curvature  of  the  lens. 

In  expressing  the  magnifying  power  of  lenses,  the  size  of  an 
object  as  seen  by  the  unaided  eye  at  ten  inches  distance  is 
taken  as  unity.  A  lens  having  a  magnifying  power  of  ten 
diameters,  or  linears,  is  one  which  enlarges  the  object  ten 
times  in  each  linear  direction. 

The  Compound  Microscope. 

The  compound,  or  ordinary,  microscope  consists  of  the 
stand  and  lenses. 

The  stand  comprises  the  following  parts  : 

1 .  Base  or  foot ; 

2.  Pillars,  or  upright,  which  may  be  jointed  or  not ; 

3.  Arm  connecting  the  pillars  with  the — 

4.  Body,  containing  the — 

5.  Draw-tube ;  moved   up  and  down  rapidly  or  slowly  by 
means  of  the — 

6.  Coarse  adjustment — used  to  bring  the  object  into  view ; 

7.  Fine  adjustment — used  only  when  the  object  is  already 
in  view,  to  bring  out  more  clearly  its  details. 


20  INTRODUCTION. 

8.  Stage — the  flat  part  on  which  is  laid  the  object  to  be 
examined,  and  which  is  perforated  by  a  central  hole  to  allow 
illumination  of  the  object  from  below.     The   stage  may  be 
circular  or  square,  stationary  or  movable,  and  mechanical. 

Underneath  the  Stage  are  Found  the  Parts: 

9.  Flat  mirror  for  low-power,  and 

10.  Concave  mirror  for  high-power  objectives. 

The  mirror  is  so  arranged  as  to  allow  motion  in  all  direc- 
tions. For  ordinary  histological  purposes  it  is  usually  fixed 
perpendicularly  to  the  stage,  and  gives  direct  light;  occasion- 
ally it  is  placed  in  an  oblique  direction,  giving  oblique  light. 

11.  Diaphragm. — Immediately  below  the  stage  and  about 
two  inches  above  the  mirror,  though  freely  movable  up  and 
down,  is  found  the  diaphragm  or  stop :  used  to  prevent  the 
peripheral  or  diffuse  rays   of   light  from    the   mirror  from 
reaching  the  object,  and  to  allow  only  the  more  central  and 
direct  rays  to  illuminate  the  same.     The  holes  in  the  dia- 
phragm are  of  different  sizes,  the  smaller  ones  being  used 
with  the  higher  power  and  the  larger  ones  with  the  lower 
power  class  of  work. 

12.  Condenser. — For  very  high  powers,  especially  such  as 
are   used  in  bacteriology,  besides  the  foregoing  parts,  there 
are  on  the  substage  the  condenser,  which  is  a  lens,  or  system 
of  lenses,  used  to  concentrate  still  further  the  light  from  the 
mirror  on  the  object.     The  condenser  most  commonly  in  use 
is  known  by  the  name  of  its  introducer  as  the  Abbe.     The 
condenser  should  be  exactly  central,  and,  as  a  rule,  it  should 
be  brought  almost  into  contact  with  the  object  on  the  stage. 

13.  Iris   Diaphragm. — Immediately  below    the   condenser, 
instead  of  the  ordinary  diaphragm,  what  is  known  as  an  iris 
diaphragm   is   used  (so  called  from   its   peculiar  variability, 
like  the  iris  of  the  eye). 

14.  Nose-piece  or  Revolver. — At  the  bottom  of   the  tube 
a  mechanical  piece,  which  enables  one  to  attach  two  or  three 
objectives  to  the  microscope  at  the  same  time,  is  known  as 
the  nose-piece  or  revolver. 


THE  MICROSCOPE.  21 

15.  Lenses. — These  are  so  important  that  a  detailed  descrip- 
tion of  them  is  necessary. 

The  Lenses  and  Lens-systems  of  the  Microscope. 

Lenses. — The  lenses  of  an  ordinary  microscope  are  of 
two  kinds :  those  attached  to  the  end  of  the  tube  nearer  the 
object,  and  known  by  the  name  of  the  objective  lens,  or 
objective  system  of  lenses,  and  those  fitting  the  end  of  the 
tube  into  which  the  observer  looks,  known  as  the  eye-piece 
or  ocular  lens. 

The  Objective  Lens  or  System  of  Lenses. 

The  objective  is  the  principal  lens  or  system  of  lenses  of 
the  microscope.  It  is  that  which  gives  the  greatest  part  of 
the  magnifying  power  to  the  instrument.  As  ordinarily 
arranged,  it  is  composed  of  a  number  of  lenses  connected 
together  in  various  ways,  and  known  as  combinations  or 
systems.  The  combination  nearest  the  object  is  called  the 
front  combination,  or  front  lens,  and  that  nearest  the  ocular 
the  back  combination,  or  back  lens.  There  may  be  one  or 
more  intermediate  systems  between  these.  Each  combination, 
or  system,  consists  of  a  concave  lens  of  flint  glass  and  a  con- 
vex lens  of  crown  glass ;  the  whole  combination  acts  as  a 
double  convex  lens.  The  purposes  of  having  lenses  of  vari- 
ous shapes  and  materials  is  to  correct  what  is  known  as 
chromatic  (colored)  and  spherical  aberration  or  distortion  (see 
Fig.  1). 

Designation  of  the  Objective. — Objectives  are  designated,  as 
a  rule,  by  their  equivalent  focal  lengths.  This  length  is  usu- 
ally given  in  inches  or  fractions  thereof — for  instance,  1  inch, 
J  inch,  T}-  inch.  In  continental  Europe  the  numerator  of  the 
fraction  is  often  omitted,  the  \  objective  being  called  3,  and 
the  ^  inch  being  called  7.  These  numbers  indicate  that  the 
objective  produces  a  real  image  of  the  same  size  as  is  pro- 
duced by  a  simple  convex  lens  whose  principal  focal  distance 
would  be  that  indicated  by  the  number.  And  as  "  the  rela- 
tive size  of  object  and  image  vary  directly  as  their  distance 


22 


INTRODUCTION. 
FIG.  1. 


— G 


Microscope :  A,  ocular  or  eye-piece ;  B,  objective  system ;  c,  stage ;  D,  iris 
diaphragm  (its  opening  may  be  diminished  or  increased  by  means  of  a  small  lever) ; 
E,  mirror  or  reflector;  F,  coarse  adjustment;  G,  fine  adjustment;  H,  substage  con- 
denser (Abbe's) ;  i,  nose-piece. 

from  the  centre  of  the  lens,"  the  less   the   equivalent  focal 
distance  of  the  objective,  the  greater  is  its  magnifying  power. 


THE  TYPES  OF  OBJECTIVE  LENS.  23 

An  objective  of  J  inch,  or  No.  3,  therefore,  magnifies  less 
than  one  of  ^  inch,  or  No.  7. 

The  working  distance  of  the  microscope — that  is,  the  dis- 
tance between  the  objective  and  the  object — is  always  less 
than  the  equivalent  focal  distance  of  the  objective. 

THE  TYPES  OF  OBJECTIVE  LENS. 

1.  Dry  and  Immersion  Objectives. 

In  the  dry  objectives,  nothing  intervenes  between  the  objec- 
tive and  the  object  to  be  examined  except  air :  all  low-power 
objectives  are  dry. 

In  the  immersion  objectives,  some  liquid,  such  as  water, 
glycerin,  or  oil  (homogeneous  immersion  objectives),  must  be 
placed  upon  the  cover-glass  over  the  object  and  make  contact 
between  the  cover-glass  and  the  objective.  Such  lenses  are 
known,  respectively,  as  water-,  glycerin-,  and  oil-immersion 
lenses.  In  homogeneous  immersion  objectives  the  oil  has  the 
same  refracting  index  as  the  front  lens  of  the  objective. 

2.  Non-achromatic  objectives  are   objectives  in   which  the 
color-distortion  is  not  corrected,  and  the  image  produced  is 
bordered  by  a  colored  fringe ;  they  also  show  spherical  dis- 
tortion. 

3.  Achromatic   objectives   are   those   in   which    the   color- 
aberration  is  corrected. 

4.  Aplanatic  objectives  are  those   in  which  the    spherical 
.aberration  is  corrected.      All  better  classes  of  objectives  are 
both  achromatic  and  aplanatic. 

5.  Apochromatic  objectives  are    objectives   in    which    rays 
of  three  spectral  colors  combine  at  one  focus  instead  of  rays 
of   two   colors,  as    in   the  ordinary  achromatic.     They   are 
highly  achromatic  objectives. 

6.  Adjustable  objectives  are  objectives  in  which  the  distance 
between  the  front  and  back  combinations  may  be  regulated 
by  means  of  a  milled-head  screw.     This  is  useful  in  dry  or 
water-immersion  objectives  to  correct  the  dispersion  of  light 
•caused  by  different  thicknesses  of  the  cover-glasses. 

The  angular  aperture  of  an  objective  is  the  angle  formed 


24  INTE  OD  UCTION. 

between  the  most  diverging  rays  issuing  from  the  axial  point 
of  an  object  that  may  enter  and  take  part  in  the  formation 
of  an  image.  By  axial  point  is  meant  a  point  situated  in  the 
extended  optical  axis  of  the  microscope. 

The  optical  axis  of  the  microscope  is  a  line  drawn  from  the 
eye  through  the  middle  of  the  tube  to  the  centre  of  the 
objective. 

Relation  of  Size  and  Working  Distance  of  the  Lens. — The 
larger  the  lens  and  the  less  its  working  distance,  the  greater 
the  angle  of  aperture.  For  dry  objectives  the  greater  the 
angular  aperture,  the  better  the  definition  of  the  objective. 

Numerical  aperture  is  the  capacity  of  an  optical  instrument 
for  receiving  rays  from  the  object  and  transmitting  them  to 
the  image,  and  the  numerical  aperture  of  a  microscopic  objec- 
tive is,  therefore,  determined  by  the  ratio  between  its  focal 
length  and  the  diameter  of  the  emergent  pencil  at  the  point 
of  its  emergence — that  is,  the  utilized  diameter  of  a  single- 
lens  objective  or  of  the  back  lens  of  a  compound  objective. 
It  is  the  ratio  of  the  diameter  of  the  emergent  pencil  to  the 
focal  length  of  the  lens,  or,  in  other  words,  it  is  the  index  of 
refraction  of  the  medium  in  front  of  the  objective  multiplied 
by  the  sine  of  half  the  aperture. 

The  Ocular  Lens  or  Eye-piece. 

The  eye-piece,  or  ocular,  is  the  lens,  or  combination  of 
lenses,  placed  in  the  tube  at  the  point  of  observation.  It 
acts  as  a  simple  microscope  and  serves  to  magnify  the 
image  of  the  object.  The  ocular  consists  of  two  lenses, 
one  situated  nearer  the  eye,  known  as  the  eye-lens,  and 
the  other  known  as  the  field-lens.  The  ocular  is  said  to 
be  positive  when  the  image  is  formed  beyond  it ;  and  nega- 
tive when  it  is  formed  within  it,  between  the  field-lens  and 
eye-lens.  In  the  positive  ocular  the  two  lenses  act  together 
as  a  simple  microscope  and  magnify  the  image.  In  the 
negative  ocular  the  field-lens  acts  with  the  objective  in 
making  clearer  the  image,  and  with  the  eye-lens  in  help- 
ing to  correct  some  of  the  aberrations.  The  eye-lens  also 
magnifies  the  image. 


THE  TYPES  OF  OCULAR  LENS.          25 

THE  TYPES  OF  OCULAE  LENS. 

1.  Compensating  oculars,  which  correct  the  chromatic  aber- 
ration of  the  ray  outside  of  the  axis. 

2.  Projecting  oculars,  used  with  the  projecting  microscope 
or  for  microphotography. 

3.  Spectroscopic  oculars. 

These  three  types,  among  many,  are  the  most  important 
and  most  frequently  employed. 

The  designation  of  oculars  is  by  their  magnifying  power 
and  equivalent  focal  distance,  and  also  by  numbers,  the  smaller 
number  designating  the  lower  power,  and  vice  versa. 

The  field  of  the  microscope  is  the  lighted  portion  which  is 
seen  when  one  looks  through  the  microscope  with  the  instru- 
ment in  focus. 

The  eye-point  is  the  distance  from  the  instrument  at  which 
the  eye  may  look  through  with  the  least  strain. 

The  Care  of  the  Microscope. 

Keep  the  instrument  cleaned,  and  see  that  all  mechanical 
parts  move  smoothly  and  evenly.  Keep  the  mirror,  con- 
denser, and  diaphragm  central — that  is,  in  the  optical  axis. 
Bring  the  object  into  view  with  the  coarse  adjustment,  and 
define  the  details  in  it  by  means  of  the  fine  adjustment.  See 
that  no  dirt  or  dust  of  any  kind  covers  the  lenses.  Should 
the  field  be  blurred  or  dim,  after  proper  focusing  and  light- 
ing, the  fault  is  either  with  the  lenses  or  the  cover-glass  is 
soiled. 

Tests  for  the  Sources  of  Dimness  in  the  Object. — By  revolv- 
ing the  ocular  with  the  eye  in  position,  the  dimness,  when 
due  to  the  ocular,  will  also  move.  By  moving  gently  the 
object  with  the  hands,  the  dimness  will  move  if  due  to  dirt 
on  the  cover-glass.  Should  the  blurring  be  stationary  in 
both  the  above  tests,  it  is  due  to  soiling  of  the  objective. 

To  cleanse  the  lenses  of  the  ocular,  blow  on  both  surfaces 
of  each  lens  and  wipe  dry  with  a  fine  silk  handkerchief,  old 
soft  linen  rag,  or,  better,  rice-paper.  To  cleanse  the  objective, 
wipe,  put  the  lens  into  the  instrument  and  test  it  as  de- 


26  INTRODUCTION. 

scribed  in  the  preceding  paragraph,  and  if  this  is  not  suffi- 
cient, pass  a  little  water  or  absolute  alcohol  over  the  surface 
and  wipe  dry.  If  the  soiling  is  due  to  balsam  or  other  resin- 
ous substance,  clean  gently  with  benzole  or  xylol.  The  back 
surface  of  the  objective  need  never  get  dirty ;  but  when  it 
does,  inserting  a  soft  rag  into  the  objective  and  gently  turn- 
ing it  around  is  sufficient  to  cleanse  it.  Never  screw  apart 
the  different  lenses  of  the  objective,  as  it  takes  an  expert  optician 
to  put  them  into  proper  position.  Always  see  that  the  cover- 
glass  is  clean  and  dry  on  its  upper  surface.  Never  bring 
the  front  lens  of  the  objective  into  direct  contact  with  the 
object  or  cover-glass. 

For  bacteriological  work,  it  is  indispensable  to  have  a 
microscope  supplied  with  an  Abbe  condenser  and  an  oil-im- 
mersion objective  of  -fa  inch  focus. 

Different  objectives  according  to  their  construction  require 
different  tube-lengths  of  the  microscope  to  magnify  at  their 
fullest  power  and  give  their  best  definition.  Manufacturers 
generally  supply  full  information  as  to  the  proper  tube-length 
for  each  instrument. 

QUESTIONS. 

What  is  refraction  ? 

Give  the  two  laws  of  refraction. 

What  is  the  type  of  the  microscope  lenses  ? 

What  is  the  focus  of  a  lens? 

What  is  meant  by  spherical  aberration  ?     How  is  this  corrected  ? 

What  are  doublets  and  triplets  ? 

What  is  meant  by  refrangibility  ?  How  is  this  corrected  in  microscope, 
lenses? 

How  many  kinds  of  microscope  are  there  ? 

What  is  a  simple  microscope  ? 

How  is  the  magnifying  power  of  lenses  expressed  ? 

What  is  meant  by  a  compound  microscope  ? 

Give  the  different  parts  of  a  compound  microscope  ? 

What  is  meant  by  direct  light  ? 

What  purpose  does  a  diaphragm  serve? 

What  is  a  condenser  ? 

What  is  meant  by  an  iris  diaphragm  ? 

What  is  a  nose-piece  ? 

How  are  the  lenses  of  an  ordinary  microscope  called  ? 

What  is  an  objective? 

How  many  lenses  or  combinations  of  lenses  does  an  ordinary  objective 
contain  ? 

How  is  the  magnifying  power  of  objectives  designated? 


THE  FUNDAMENTAL  PRINCIPLES.  27 

What  is  meant  by  the  focal  distance  of  an  objective?  The  working  dis- 
tance? 

What  is  the  difference  between  the  dry  and  immersion  objectives? 

What  is  a  homogeneous  immersion  objective? 

What  is  meant  by  a  non-achromatic  objective? 

What  is  meant  by  an  achromatic  objective?    An  aplanatic  objective  ? 

What  is  an  apochromatic  objective? 

What  is  meant  by  an  adjustable  objective? 

What  is  the  angular  aperture  of  an  objective? 

What  is  meant  by  the  actual  point  of  an  objective? 

What  is  the  optical  axis  of  a  microscope  ? 

What  relation  does  the  size  of  the  lenses  have  to  its  angular  aperture? 

What  is  the  numerical  aperture  of  an  objective  ?  What  is  the  ocular  of  a 
microscope  ? 

Of  how  many  lenses  does  it  consist  ? 

What  is  the  difference  between  the  positive  and  the  negative  ocular. 

What  is  a  compensating  ocular?    A  projecting? 

How  are  oculars  designated  ? 

What  is  the  field  of  a  microscope  ? 

What  is  the  eye-point? 

What  care  should  be  given  to  a  microscope? 

Describe  the  tests  for  determining  the  cause  of  an  obscure  image. 

Describe  the  methods  of  cleansing  the  lenses  of  the  microscope. 


CHAPTER  I. 
THE  FUNDAMENTAL  PRINCIPLES. 
THE  HISTORY  OF  BACTERIOLOGY. 

in  the  latter  part  of  the  seventeenth  century 
Anthony  von  Leuwenhoek,  by  means  of  his  magnify  ing- 
glasses,  first  discovered  organisms  in  decaying  vegetable 
infusions,  he  may  be  said  to  have  laid  the  very  first  stone  in 
the  foundation  of  what  later  on  was  to  be  the  Science  of 
Bacteriology. 

It  was  very  long  after  this,  however,  before  sufficient  facts 
were  collected  to  place  this  science  upon  a  firm  basis,  and  it 
remained  for  a  genius  like  the  immortal  Pasteur  and  the 
eminent  talents  of  the  equally  great  Koch  to  build  up  the 
superstructure  of  bacteriology  so  as  to  have  it  accepted  by 
all  as  the  true  basis  of  scientific  medicine. 

When  first  observed,  these  microorganisms  were  supposed 


28  THE  FUNDAMENTAL  PRINCIPLES. 

to  be  animalcules,  and  were  accepted  as  such  until  the  middle 
of  the  nineteenth  century,  when  F.  Cohn  classed  them  as 
belonging^  to  the  vegetable  kingdom,  and  listed  them  among 
the  fungi,  making  of  them  the  third  variety  of  fungi,  the 
schizomycetes  or  cleft  fungi ;  the  other  two  being  the  saccharo- 
mycetes  or  sprouting  fungi  (the  yeast  plant),  and  the  hyphomy- 
cetes  or  mucorini  (the  moulds). 

THE  CLASSIFICATION  OF  COHN  FOR  BACTERIA. 

This,  as  just  given,  is  accepted  to-day  by  all  authori- 
ties, though  it  is  open  to  criticism.  Although  it  is  true  that 
the  great  majority  of  these  organisms  like  the  fungi  possess 
no  chlorophyl,  and  are  unable,  like  other  vegetables,  to  obtain 
their  nourishment  from  the  carbon  dioxide  and  nitrogen  of 
the  atmosphere,  but,  on  the  contrary,  like  animals,  require 
higher  carbohydrate  and  nitrogenous  substances,  which  they 
decompose  into  their  primitive  elements  for  their  subsistence. 
A  few  of  them,  however,  possess  some  plant  coloring-matter, 
and  some  seem  able  to  thrive  in  a  simple  saline  solution  from 
which  absolutely  no  nitrogen  is  to  be  obtained. 

THE  DEFINITION  OF  "  BACTERIA." 

The  proper  name  therefore  for  these  organisms,  and  the 
one  generally  adopted,  is  bacteria,  which  is  the  plural  of  the 
Latin  substantive  bacterium.  They  may  be  defined  as  fol- 
lows :  Unicellular  vegetables  of  low  organization,  devoid  of 
chlorophyl  (plant  color  ing -matter),  and  multiplying  by  fission. 

The  bacteria  cells  consist  of  a  cell-membrane  and  protoplasm, 
which  latter  is  sometimes  clear  and  sometimes  granular,  but 
with  no  nuclei.  The  cell-membrane  is  a  firm,  tough  envelope, 
very  much  like  cellulose,  which  occasionally  in  some  bacteria 
becomes  viscid  and  gelatinous  in  its  outer  layers,  forming  a  sort 
of  bright  halo  around  the  bacteria,  called  a  capsule.  This 
gelatinous  matter  occasionally  serves  to  bind  two  or  more 
bacteria  together,  and  gives  to  them  quite  a  characteristic 
grouping  which  helps  to  distinguish  them  from  others.  In 
some  instances  the  membranous  envelope  interferes  consid- 


MORPHOLOGICAL  CLASSIFICATION  OF  BACTERIA.     29 

erably  with  the  staining  of  the  protoplasm  of  the  bacteria 
cells,  so  that  special  methods  of  staining  have  to  be  adopted 
for  these.  Again,  those  bacteria  which  are  generally  found 
surrounded  by  a  capsule,  when  grown  in  artificial  media  seem 
to  lose  the  power  of  developing  capsules. 


THE  MORPHOLOGICAL  CLASSIFICATION  OF  BACTERIA. 

Bacteria  are  divided  into  three  varieties :  (1)  the  rounded 
form  or  coccus  (plural  cocci) ;  (2)  the  rod-shaped  form  or 
bacillus  (plural  bacilli) ;  and  (3)  the  curved  or  spiral  form, 
spirillum  (plural  spirilla). 

I.  The  Coccus. 

Varieties. — The  cocci,  which  are  not  always  round,  but 
very  often  oval  in  form,  are  further  distinguished  according 
as  they  appear :  singly  and  of  large  size,  as  megacocci;  of 
small  size,  as  micrococci;  double — that  is,  two  of  the  cells 
adhering  together,  as  diplococci ;  in  chains — that  is,  a  number 
of  cells  adhering  together  in  single  file — as  streptococci;  in 
groups  very  like  a  bunch  of  grapes,  as  staphylococci ;  in 
groups  of  four,  as  tetrads  or  merismopedia ;  in  groups  of  eight 
arranged  in  cubes,  as  sarcinae ;  in  irregular  masses  united  by 
an  intercellular  substance  and  imbedded  in  a  tough  gelati- 
nous matrix,  as  ascococci. 

II.  The  Bacillus. 

Morphology. — The  bacilli  or  rod-shaped  (desmo-)  bacteria 
are  distinguished  by  the  fact  that  their  two  longest  sides  are 
parallel  to  each  other ;  the  two  short  sides  being  at  times 
straight,  at  others  concave,  and  at  others  again  convex. 

Varieties. — They  are  said  to  be  (1)  slender  when  their 
breadth  is  to  their  length  as  1  to  4  or  more,  and  (2)  thick 
when  it  is  as  1  to  2.  They  develop  singly  or  in  pairs  or  in 
long  threads  or  filaments,  being  attached  together  always  by 
their  narrow  ends. 


30 


THE  FUNDAMENTAL  PRINCIPLES. 


III.  The   Spirillum. 

The   spirilla   or   curved  or   spiral  bacteria  develop  either 
singly  or  in  pairs  or  in  long  twisted  or  corkscrew  filaments. 

The  Variations  in  Development  of  Each  Species. 
Though  under  varied  conditions  of  growth  the  form  of 
any  one  species  may  vary  considerably,  yet  these  three  main 
divisions  under  similar  conditions  are  permanent — that  is, 
micrococci  always  develop  into  micrococci,  bacilli  into  bacilli, 
and  spirilla  into  spirilla. 

FIG.  2. 


00    o»  <*> , 
*       d» 


e  a  e 

a.  Staphylococci    b.  Streptococci,    c.  Diplococci.    d.  Tetrads,    e.  Sarcinse.    (Abbott.) 

FIG.  3. 


Diplococcus  of  pneumonia,  with  surrounding  capsule.    (Park.) 


MORPHOLOGICAL  CLASSIFICATION  OF  BACTERIA.     31 
FIG.  4. 


\ 


<Cr- 


''' 


d  e  f 

a.  Bacilli  in  pairs.    6.  Single  bacilli,    c  and  d.  Bacilli  in  threads,    e  and  /. 

Bacilli  of  variable  morphology.    (Abbott.) 


FIG.  5. 

%   «*• 

,^v~ 

^TV/^  „  ,TM 

«  6  c  ^ 

a  and'd  Spirilla  in  short  segments  and  longer  threads— the  so-called  comma 
f9rms  and  spirals,  b.  The  forms  known  as  spirochseta.  c.  The  thick  spirals  some- 
times known  as  vibrios.  (Abbott.) 


FIG.  6. 


U  It 

a.  Spirillum  of  Asiatic  cholera  (comma  bacillus) ;  normal  appearance  in  fresh  culf 
ures.    b.  Involution-forms  of  this  organism  as  seen  in  old  cultures.    (Abbott.) 


Occasionally  under  peculiar  conditions  what  are  known  as 
involution-forms  are  produced,  forms  which  may  scarcely  be 
recognized  as  those  belonging  to  the  original  bacteria.  These 
points  are  shown  by  Figs.  2,  3,  4,  5,  6. 


32  THE  FUNDAMENTAL  PRINCIPLES. 

THE  SIZE  OF  BACTERIA. 

Bacteria  require  to  be  seen  and  studied  by  the  highest  powers 
of  the  microscope ;  they  vary  in  size  from  0.2  to  30  mikrons. 
(A  mikron  isyTJW  millimeter;  about  2"5iro"o  illcn«)  The  micro- 
cocci  have  a  diameter  of  from  0.2  to  1  mikron  or  more.  Bacilli 
and  spirilla  vary  in  length  from  2  to  30  mikrons  or  more ; 
in  breadth,  from  1  to  4  mikrons.  The  average  length  of 
pathogenic  bacilli  is  3  mikrons. 

THE  REPRODUCTION  OF  BACTERIA. 

As  mentioned  in  the  foregoing  paragraphs,  bacteria  multi- 
ply by  fission. 

I.  Fission. 

In  the  case  of  cocci,  the  round  or  oval  cells  show  a  little 
indentation  beginning  in  the  membrane  at  two,  four,  or  eight 
points  of  its  periphery,  according  as  the  division  is  to  occur 
in  two,  four,  or  eight  parts ;  this  indentation  increases  until 
the  original  cell  is  divided  into  hemispheres,  quadrants,  or 
octants,  as  the  case  may  be.  These  parts  remain  attached  to 
one  another  until  complete  spheric  cocci  are  formed  from 
each  part,  and  they  then  separate  or  not  according  to  the 
nature  of  the  bacteria. 

In  some  forms  of  diplococci,  as  the  gonococci,  complete 
spheres  are  never  formed,  the  cells  remaining  attached  to 
each  other  in  pairs  as  hemispheres. 

In  the  case  of  nearly  all  bacilli  and  spirilla  the  cells  increase 
to  nearly  double  their  original  size  before  division,  and  the 
division  always  takes  place  in  the  direction  of  the  length  of  the 
bacterium.  The  daughter  cell  remains  attached  for  a  while 
to  its  parent  cell  after  fission  is  complete  ;  occasionally  this 
attachment  persists  for  a  long  time,  so  that  large  filaments 
consisting  of  a  number  of  bacteria  are  formed. 

II.  S population. 
1.   The  Endospore. 

Division  by  fission  is  the  usual  mode  of  the  reproduction 
of  bacteria,  but  at  times,  depending  upon  various  circum- 


THE  REPRODUCTION  OF  BACTERIA.  33 

stances  to  be  mentioned  later,  what  are  known  as  spores  are 
formed  by  a  number  of  bacilli  and  spirilla.  These  spores, 
which  are  perhaps  the  equivalents  of  seeds  for  the  higher 
plants,  are  formed  in  this  way  :  in  the  body  of  the  bacillus, 
generally  at  its  centre,  occasionally  at  one  of  its  poles,  a 
number  of  dark  highly  refractile  granules  accumulate,  and 
are  soon  changed  into  an  oval,  glistening,  highly  refractile 
body  which  is  surrounded  by  a  membrane  of  the  same  com- 
position as  that  of  the  cell  itself,  but  thicker  and  more 
resistant.  One  spore  only  is  formed  in  each  cell.  Some- 
times the  spore-formation  causes  no  change  in  the  shape  of 
the  rod.  At  other  times  there  is  a  bulging  of  the  centre  of 
the  body  of  the  bacillus,  where  the  spore  is  located,  with  a 
general  tapering  to  the  two  ends,  giving  to  the  bacillus  the 
shape  of  a  spindle.  This  is  called  a  clostridium.  Again, 
when  the  spore  is  formed  at  one  of  the  poles,  there  is  some- 
times a  bulging  of  that  part,  giving  to  the  bacillus  the 
appearance  of  a  nail  or  drum-stick,  whence  the  name  of  drum- 
mer-bacillus for  the  cell.  Fig.  7  shows  these  forms  well. 


Fio.  7. 


\  N> 


<a.  Bacillus  suUilis  with  spores,    b.  Bacillus  anthracis  with  spores,   c.  (Jlostridiumfonn 
with  spores,    d.  Bacillus  of  tetanus  with  endospores.    (Abbott.) 

Soon  after  the  formation  of  the  spore  the  rest  of  the  body 
of  the  cell  disintegrates  and  breaks  down,  and  the  oval  spore 
is  liberated. 

The  spores  are  characterized,  on  account  of  their  thick 
membrane,  by  resistance  to  external  influences  which  would 
be  fatal  to  the  bacilli  themselves,  such,  for  instance,  as 
extremes  of  heat  or  cold,  desiccation,  and  the  action  of  chemi- 
cals;  also,  to  a  great  extent,  staining  by  the  penetration  into 
3— M.  B. 


34  THE  FUNDAMENTAL  PRINCIPLES. 

their  body  of  certain  dyes  which  have  great  affinity  for  the 
protoplasm  of  ordinary  bacteria,  so  that  a  special  method 
must  be  adopted  for  their  staining,  as  will  be  described 
later. 

Spores  therefore  preserve  the  species,  when  these  would  be 
destroyed  if  dependent  solely  on  the  bacilli  for  their  preser- 
vation. 

2.  The  Arthrospore. 

The  foregoing  spore-formation,  known  as  endospores,  is  the 
usual  mode  of  spore-formation  found  in  bacteria,  and  is  lim- 
ited to  the  rod  and  spirilla  forms ;  but  another  form  of 
spore,  called  arthrospore,  is  mentioned  by  some  as  occurring 
occasionally  in  the  round  or  cocci  forms.  This  consists  in  a 
special  jointed  projection  forming  from  the  outside  of  the 
cells,  and  capable  later  of  developing  into  the  original  cell. 
This  form  of  spore-formation  is  generally  doubted  at  the  present 
time. 

Spores  are  incapable  of  producing  other  spores,  and  can,  only 
when  placed  in  suitable  conditions,  develop  into  the  type  of 
bacilli  which  gave  them  birth.  When  this  occurs  the  spore 
begins  to  elongate,  loses  its  glistening  appearance,  and  finally 
its  membrane  ruptures  at  one  end  or  in  the  centre  and  gives 
exit  to  a  fully  developed  bacillus. 

Spores  may  be  distinguished  from  rounded  bacteria  by 
means  of  their  brighter,  more  glistening  appearance,  their 
power  of  resisting  stains,  and  the  fact  that  in  suitable  media 
they  develop  into  bacilli. 

Significance  of  Sporulation. — Whether  the  original  idea  that 
spores  form,  in  bacteria  capable  of  producing  them,  only 
when  the  latter  are  submitted  to  external  noxious  influences, 
and  for  the  purpose  of  perpetuating  the  species,  or  whether, 
as  more  recently  maintained,  sporulation  is  the  result  of  the 
highest  expression  of  the  complete  development  of  bacteria, 
is  not  at  present  fully  determined,  though  the  dictum  of 
authorities  inclines  to  the  latter  view,  and  the  spore-formation 
of  some  of  the  best-known  and  studied  bacteria,  as  anthrax, 
seems  to  lend  color  to  this  theory. 


THE  MOTILITY  OF  BACTERIA.  35 

THE  MOTILITY  OF  BACTERIA. 

Motility,  or  the  power  of  transporting  themselves  from 
place  to  place,  is  possessed  by  a  number  of  bacteria.  This 
is  effected  by  filamentous  or  hair-like  processes  arising  from 
the  body  of  the  bacteria,  and  called  flagella.  It  must  not 
be  confounded  with  the  peculiar  whirling  or  dancing  move- 
ment so  often  seen  under  the  microscope,  even  in  inorganic 
particles,  and  called  the  Brownian  movements.  Motility, 
except  in  two  instances,  has  so  far  been  observed  only  in 
bacilli  and  spirilla.  Its  rapidity  depends  on  the  particular 
bacterium,  its  mode  of  cultivation,  the  age  of  the  culture, 
and  other  similar  factors.  Some  bacteria  after  repeated  arti- 
ficial cultivation  seem  to  lose  their  motility,  which,  however, 
may  be  restored  fully  by  passing  through  an  animal. 

FIG.  8. 


a.  Spiral  forms  with  a  flagellum  at  only  one  end.  6.  Bacillus  of  typhoid  fever 
with  flagella  given  off  from  all  sides,  c.  Large  spirals  from  stagnant  water  with 
wisps  of  flagella  at  their  ends  (Spirillum  undula).  (Abbott.) 

The  flagella  are  hair-like  processes  consisting  of  the  same 
material  as  the  bacterial  cell-membrane,  and  are  so  minute  as 
scarcely  to  be  visible  under  the  highest  power  of  the  micro- 
scope unless  they  are  stained  by  special  processes,  as  will  be 
described  in  the  chapter  on  staining.  Whenever  there  is 
only  a  single  flagellum  at  one  of  the  poles  of  the  bacillus,  this 
is  said  to  be  monotrocha  ;  whenever  there  is  a  single  flagellum 
at  each  pole  of  the  bacterium,  it  is  said  to  be  amphitrocha ; 
whenever  there  is  a  cluster  of  flagella  at  one  pole,  the  bac- 
terium is  said  to  be  lophotrocha ;  and  finally,  when  a  varying 
number  of  flagella  seem  to  arise  from  different  portions  of 
the  body  of  the  bacterium,  it  is  said  to  be  peritrocha.  These 
features  are  shown  in  Fig.  8. 


36  THE  FUNDAMENTAL  PRINCIPLES. 

THE  RELATION  OF  OXYGEN  TO  BACTERIAL  LIFE. 

Bacteria  are  divided  into  aerobic  and  anaerobic  according 
as  they  require  oxygen  or  not  for  their  development.  Some 
thriving  best  in  the  presence  of  oxygen  are  able  to  develop, 
however,  without  the  presence  of  this  gas ;  these  are  called 
facultative  anaerobic.  Others  thriving  best  without  oxygen 
but  able  to  develop  in  the  presence  of  this  gas  are  called 
facultative  aerobic. 

THE  RELATION  OF  DEAD  AND  LIVING  ORGANIC  MATTER 
TO  BACTERIA. 

Bacteria  are  also  divided  into  saprophytes  and  parasites, 
according  as  they  require  for  their  development  merely  the 
presence  of  decomposable  organic  matter  or  the  body  of  a 
living  higher  organism,  as  host,  on  which  they  live.  Should 
they  also  be  able  to  live  outside  their  host,  they  are  called 
facultative  parasites.  The  disease-producing  germs  are  always 
parasitic. 

THE  ESSENTIAL  CONDITIONS  OF  BACTERIAL  GROWTH. 

For  the  proper  development  of  bacteria  the  following  are 
required :  heat,  moisture,  and  the  presence  of  some  decom- 
posable organic  matter,  with  special  chemical  reaction  of  the 
culture-medium. 

I.  Heat. 

A  temperature  between  10°  and  40°  C.  is  required  for  the 
development  of  adult  bacteria,  but  the  degree  varies  greatly 
according  to  the  different  species.  Some  of  the  parasites 
thrive  best  at  a  temperature  in  the  neighborhood  of  that  of 
the  human  body,  about  36°  or  37°  0. ;  others,  the  saprophytes, 
seem  to  grow  best  at  the  ordinary  room  temperature,  between 
20°  and  30°  C.  ;  some  few  again  seem  to  be  able  to  grow 
and  multiply  at  a  temperature  near  the  freezing-point ;  and 
not  long  ago  a  number  have  been  shown  to  develop  abun- 
dantly at  a  temperature  above  70°  C.  As  a  general  rule, 
however,  a  temperature  of  0°  C.  and  one  above  60°  C.  are  fatal 


ESSENTIAL  CONDITIONS  OF  BACTERIAL   GROWTH.    37 

within  a  few  minutes  for  the  ordinary  non- spore-beating  bacteria. 
Spores  are  able  to  resist  for  a  long  time  the  effects  of  cold  and 
excessive  heat.  Some  have  developed  after  having  been  im- 
mersed for  a  long  time  in  liquid  air  (temperature,  — 200°  C.), 
and  also  after  exposure  to  dry  heat  of  above  150°  F.  for 
sixty  minutes  and  moist  heat  for  thirty  or  forty  minutes. 

II.  Moisture. 

A  certain  amount  of  moisture  is  absolutely  indispensable 
for  the  growth  of  bacteria,  desiccation  being  fatal  within  a 
few  minutes  for  nearly  all  the  fully  formed  bacteria. 

Spores,  however,  are  capable  of  developing  after  being  kept 
dry  for  an  indefinite  period. 

III.   Decomposable   Organic  Material. 

A  certain  amount  of  decomposable  organic  matter  is  indis- 
pensable for  the  development  of  the  bacteria.  This  they 
decompose  into  simple  elements,  and  are  so  able  to  obtain  the 
nitrogen  and  carbon  necessary  for  their  sustenance.  At  the 
same  time  they  set  free  carbon  dioxide  and  nitrogen  from  the 
remainder,  and  so  provide  for  the  nourishment  of  the  higher 
vegetables,  which  must  have  these  substances  free  in  order  to 
support  life.  And  in  so  doing  bacteria  render  a  service  of 
incalculable  value,  as  without  it  life  in  the  animal  kingdom 
would  soon  be  extinct. 

Different  bacteria  require  a  greater  or  lesser  proportion  of 
the  proteid  substances  for  their  nutrition ;  and  according  as 
substances  contain  these  in  a  more  or  less  favorable  condition 
for  absorption  they  are  said  to  be  more  or  less  good  culture- 
soils  or  media. 

Some  few  species  seem  to  be  able  to  live  in  saline  solution 
and  in  other  media  where  there  is  no  appreciable  amount  of 
organic  matter,  but  in  these  cases  they  probably  obtain  their 
nutrition  from  the  decomposition  of  slight  traces  of  ammonia 
and  the  carbon  dioxide  of  the  air  contained  in  the  water. 

Again,  some  bacteria  seem  to  have  the  power  of  decomposing 


38  THE  FUNDAMENTAL  PRINCfPLES. 

ammonia  and  building  up  nitrite  and  nitrate  compounds  ;  these 
are  known  as  the  nitrifying  bacteria,  and  as  a  class  are  im- 
portant, and  are  being  carefully  studied. 

IV.   Special  Chemical  Reaction  of  the  Culture -medium. 

Bacteria  are  likewise  profoundly  influenced  in  their  growth 
by  the  reaction  of  the  medium  in  which  they  grow,  most  bac- 
teria requiring  a  neutral  or  faintly  alkaline  medium,  some  few 
a  faintly  acid  one.  This  characteristic  is  found  an  element 
of  danger  for  bacterial  life  :  for  the  bacteria  which  require 
alkaline  surroundings  have,  as  a  rule,  the  property  of  secret- 
ing acids,  so  that  after  a  while  they  render  the  medium  unfit 
for  their  own  further  growth  long  before  the  pabulum  is 
exhausted. 

THE  INERT  AND  INHIBITIVE  CONDITIONS  OF  BACTERIAL 

LIFE. 

1.  Diffuse  daylight  seems  to  have  little  or  no  influence  on 
bacterial  growth,  but  most  bacteria  and  their  spores  are  killed 
after  a  more  or  less  prolonged  exposure  to  the  direct  rays  of 
the  sun,  a  fact  of  great  importance  in  practical  hygiene. 

2.  Electric  currents  and  the  X-rays  seem  to  have  but  little 
influence  on  bacterial  growth. 

3.  Compressed  air  seems  to  retard  the  growth  of  bacteria. 

4.  A  number  of  chemical  substances  either  kill  off  bacteria 
or  arrest  their  growth — as  will  be  spoken  of  more  fully  under 
the  head  of  Antiseptics  and  Disinfectants. 

THE  VITAL  MANIFESTATIONS  OR  FUNCTIONS  OF 
BACTERIA. 

These  are  manifold  and  various,  and  occasionally  attempts 
at  classification  have  been  based  on  some  of  these  special 
functions. 

1.  Fermentation. — The  alcoholic  and  acetic  acid  fermentation 
are  the  work  of  the  yeast  fungi  ;  but  the  butyric  and  lactic 
acid  fermentation  in  milk  are  caused  by  a  number  of  bacteria, 


VITAL  MANIFESTATIONS  OR  FUNCTIONS  OF  BACTERIA.   39 

the  most  prominent  of  which  are  those  that  bear  the  respec- 
tive names.     This  class  of  bacteria  are  called  the  zymogenic. 

2.  Putrefaction  is  caused  by  a  variety  of  bacteria  called 
saprogenic. 

3.  Pigments  and  colors  are  produced  by  a  number  of  bacteria 
called  chromogenic.     Sometimes  the  pigment  is  secreted  by  the 
cells   and   diffused   in    the  surrounding  media,  the   bacteria 
remaining  uncolored.     At  other  times  the  pigment  is  limited 
to  the  cell-protoplasm  and  membrane,  the  surrounding  medium 
remaining  uncolored.     As  a  rule,  these  pigments  are  produced 
only  in  an  atmosphere  of  oxygen. 

4.  Some    bacteria,    called    photogenic,   phosphorescent,    or 
fluorescent,  have  the  property  of  emitting  and  producing  light. 

5.  Bacteria  secrete  poisonous   substances  which  are  some- 
times   highly  deadly,  and   which   from    their  character   are 
classed  as  ptomaines  and  toxalbumins. 

The  ptomaines  are  crystallizable  basic  substances,  closely 
allied  to  the  vegetable  alkaloids. 

The  toxalbumins  are  non-crystallizable  substances,  similar 
to  albumin  or  protein. 

It  is  by  the  poisonous  effects  of  these  toxins  that  most 
bacteria  affect  the  human  body. 

6.  Some  bacteria  liquefy  gelatin,  and  this  fact  is  made  use 
of  in  differentiating  the  different  species.     This  liquefaction 
has  been  demonstrated  to  be  caused  by  a  soluble  peptonizing 
ferment  secreted  by  the  bacteria  cells,  and  after  filtration  of 
a  bouillon  culture  of  the  liquefying  bacterium  the  filtrate  free 
from  bacteria  possesses  the  same  power  of  liquefying  gelatin. 

7.  Some  bacteria  produce  acids  and  other  alkalies — as  may 
be  demonstrated  by  their  action. 

8.  Various  gases,  such  as  carbon  dioxide,  marsh  gas,  hydro- 
gen   sulphide,  etc.,  are   the   products  in  some  instances  of 
bacterial  growth. 

9.  A  number  of  bacteria  produce  odors  without  the  apparent 
production  of  gases. 

10.  A  few  produce  aromatics,  and  are  used  extensively  in 
the  arts. 

11.  Again,  a  certain  class   peptonize   milk   by  means   of 


40  THE  FUNDAMENTAL  PRINCIPLES. 

enzymes  which  they  secrete.     Some  cause  a  reduction  of  the 
nitrites. 

12.  Others  oxidize  nitrogen  into  nitrites,  and  even  nitrates. 

13.  Finally,  some  bacteria  cause  disease  in  man  and  animals. 
These,    called   pathogenic,    are  of    the    greatest   interest   to 
physicians,  and  it  is  the  discovery  of  this  pathogenic  prop- 
erty which   in    the   last  twenty  years    has    given    such   an 
impetus  to  the  study  of  bacteriology. 

QUESTIONS. 

Who  first  discovered  microorganisms  iu  decaying  vegetable  infusions  ? 

To  what  kingdom  were  they  thought  to  belong  at  first  ? 

What  classification  is  generally  accepted  at  present  ? 

Name  the  three  varieties  of  fungi.  What  criticism  may  be  made  of  this 
classification  ? 

Give  a  proper  definition  of  bacteria. 

Of  what  does  the  bacteria  cell  consist? 

What  causes  some  bacteria  to  have  a  capsule?  Are  the  capsules  generally 
retained  by  bacteria  when  grown  artificially  ? 

What  causes  some  bacteria  to  be  more  difficult  of  staining  than  others? 

Into  what  three  varieties  or  species  are  bacteria  divided  ? 

What  are  megacocci  ?  Micrococci  ?  Diplococci  ?  Merismopedia  ?  Sarcinse  ? 
Streptococci  ?  Staphylococci  ?  Ascococci  ? 

What  is  the  shape  of  bacilli?  When  are  they  said  to  be  slender?  When 
thick  ?  How  do  they  develop  ?  What  are  spirilla  ?  Can  one  species  develop 
into  another  species? 

What  are  the  limits  of  size  of  bacteria  ? 

What  is  a  mikron  ? 

What  is  the  average  length  of  the  pathogenic  bacteria  ? 

How  do  bacteria  multiply  ?  Describe  this  process  in  the  case  of  cocci, 
bacilli,  and  spirilla? 

What  are  spores,  and  how  are  they  formed  ? 

How  do  spores  compare  with  bacteria  in  their  power  of  resisting  injurious 
external  influences  and  of  staining  ?  To  what  is  this  due  ? 

What  is  meant  by  a  clostridium  ? 

What  is  meant  by  a  drummer-bacillus? 

What  is  the  name  of  the  ordinary  spores? 

What  is  meant  by  arthrospores  ? 

What  is  the  theory  of  spore-formation  ? 

What  is  meant  by  motility,  and  what  species  have  this  power? 

What  are  flagella  ? 

What  names  are  given  to  bacteria  according  to  the  position  and  number 
of  their  flagella? 

What  is  meant  by  aerobic  bacteria  ?  Anaerobic  ?  Facultative  aerobic  ? 
Facultative  anaerobic? 

What  are  saprophytes?    Parasites ? 

What  conditions  are  required  for  the  proper  development  of  bacteria? 

What  is  the  most  favorable  temperature  for  bacterial  growth  ? 

What  is  the  effect  of  cold  ?    Of  excessive  heat  ? 

How  do  spores  difler  from  bacteria  in  their  reaction  to  heat  and  cold  ? 


EXAMINATION  AND  STAINING   OF  BACTERIA.         41 

What  is  the  effect  of  moisture  on  bacterial  life  ?    Of  desiccation  ? 
What  pabulum  is  necessary  for  the  life  of  bacteria? 

How  do  you  explain  the  life  of  bacteria  in  saline  solution  and  in  media 
with  no  appreciable  amount  of  organic  matter  ? 
What  is  meant  by  culture-soils  or  media  ? 
What  other  conditions  influence  bacterial  growth  ? 

What  is  the  effect  of  sun-light  on  bacteria  ?    Of  electric  currents  ?    X-rays  ? 
( )f  compressed  air? 

What  are  the  manifold  functions  of  bacteria  ?    Enumerate  and  explain 
the  same. 

What  is  the  chemical  difference  between  a  ptomaine  and  a  toxalbumin  ? 
What  are  zy mogenic  ?  Chroniogenic  ?  Photogenic  ?  Pathogenic  bacteria  ? 


CHAPTER    II. 

THE  EXAMINATION  AND  THE  STAINING  OF  BACTERIA. 

THE  EXAMINATION  OF  BACTERIA. 

FOR  the  purpose  of  examining  bacteria  the  highest  power 
of  the  microscope  is  necessary,  although  many  are  seen  with  an 
ordinary  dry  ^  or  ^  objective.  Ordinarily,  however,  the  -^ 
oil-immersion  objective  is  indispensable  for  the  proper  study 
of  microorganisms. 

Bacteria  are  examined  either  alive  and  in  their  natural  con- 
dition, or  dried  on  microscope  slides  or  cover-glasses  as  a 
thin  film,  and  stained. 

1.  For  the  purpose  of  examining  bacteria  in  their  natural 
condition,  it  is  only  necessary,  when  the  bacteria  are  in  liquid 
medium,  to  put  a  droplet  of  the  liquid  on  a  slide,  cover  lightly 
with  a  thin  cover-glass,  put  upon  the  stage  of  the  microscope, 
and  bring  into  focus  the  -fa  inch  oil-immersion  objective, 
being  careful  to  close  almost  completely  the  iris  diaphragm 
underneath  the  substage  condenser. 

When  the  bacteria  are  in  solid  medium,  a  minute  particle  of 
the  culture  is  taken  up  on  a  sterilized  platinum  needle  and 
stirred  up  in  a  small  drop  of  sterilized  water  on  a  slide,  and  a 
cover-glass  applied  as  above. 

In  these  ways  the  form,  shape,  mode  of  grouping,  and  motility 


42        EXAMINATION  AND  STAINING   OF  BACTERIA. 

of  the  bacteria  may   be    very  well  seen  and    carefully   in- 
vestigated. 

The  Hanging-drop  Preparation. 

When  it  is  necessary  to  keep  them  under  observation  for 
some  time,  however,  or  when  it  is  desired  to  study  their 
development,  multiplication,  or  sporulation,  what  is  known  as 
the  hanging-drop  preparation  or  culture  is  resorted  to. 

This  consists  in  placing  a  small  drop  of  the  liquid  contain- 
ing the  bacteria  on  a  thin  cover-glass  and  placing  upon  this 
cover-glass  a  slide  with  a  concavity  in  its  centre,  known  as 
the  hanging-drop  slide,  after  having  carefully  lubricated  the 
edges  of  the  surface  around  the  concavity  with  vaselin,  so 
that  the  slide  will  adhere  to  the  cover-glass  when  it  is  pressed 
down  on  it.  In  this  way  a  hermetically  sealed,  transparent, 
moist  chamber  is  obtained,  which  may  be  kept  under  observa- 
tion on  the  stage  of  a  microscope  almost  indefinitely. 


Longitudinal  section  of  hollow  ground  glass  slide  for  observing  bacteria  in 
hanging  drops.     (Abbott.) 

2.  The  foregoing  are  very  simple  and  useful  methods  for 
rapid  examinations  of  bacteria,  and  have  many  applications, 
but  they  are  far  from  satisfactory  in  all  cases,  as  they  fail  to 
bring  out  full  details  of  bacterial  structure.  For  this  purpose 
recourse  must  be  had  to  the  staining  methods  introduced  by 
Koch,  and  perfected  by  Weigert,  Loeffler.,  and  many  others. 
In  this  method  bacteria  are  examined  dead. 


THE  STAINING  OF  BACTERIA. 

I.   The   General  Mode   of  Procedure, 

One  or  several  droplets  of  the  suspected  liquid  are  spread 
thinly  and  evenly  on  the  surface  of  a  slide  or  thin  cover-glass, 


THE  STAINING   OF  BACTERIA.  43 

or,  in  case  of  a  solid,  a  small  particle  is  diluted  with  sterile 
water  and  spread  in  the  same  way  on  the  slide  or  cover-glass. 
This  is  allowed  to  dry  in  the  air,  protected  from  dust,  or  else 
is  held  in  a  suitable  forceps  high  up  over  the  flame  of  an 
alcohol  lamp  or  Bunsen  gas-burner  until  dry,  thus  forming  a 
thin  film  on  the  surface  of  the  glass.  After  this  step  has 
been  carefully  taken  the  slide  or  cover-glass  is  taken  up  with 
a  pair  of  forceps  and  passed  several  times  through  the  flame, 
film-side  up,  for  one-half  to  one  second  at  each  pass,  three- 
times  in  the  case  of  a  cover-glass  and  eight  or  ten  passes  in  the 
case  of  a  slide;  which  is  for  the  purpose  of  setting  the  prepa- 
ration, or  coagulating  the  albuminoids  and  fixing  them  to  the 
glass  so  that  they  will  not  be  easily  washed  away  in  the  sub- 
sequent procedures. 

After  allowing  the  preparation  to  cool  well,  it  is  ready  for 
the  staining  reagents. 

II.  The  Most  Commonly  Used  Stains. 

The  basic  anilin  dyes,  such  as  fuchsin,  methylene-blue, 
gentian-violet  or  methyl-violet,  and  Bismarck-brown,  are 
most  commonly  employed. 

They  are  made  into  saturated  alcoholic  solutions,  to  be  kept 
in  stock,  and  are  freely  diluted  with  water  whenever  required 
for  use. 

III.  The  Application  of  the  Dyes. 

Nearly  all  the  known  bacteria,  with  the  exceptions  to  be 
mentioned  later,  are  readily  stained  by  the  watery  solutions 
of  any  of  the  basic  anilin  dyes.  The  film  on  the  slide  or 
cover-glass,  prepared  as  just  described,  is  covered  by  a  few 
drops  of  the  stain,  or  the  cover  glass,  film-side  down,  is  floated 
in  a  watch-glass  full  of  the  staining  solution;  at  the  end  of 
from  one-half  to  two  or  three  minutes  the  staining  fluid  is 
poured  off,  the  slide  or  cover-glass  washed  rapidly  in  water 
and  then  allowed  to  air-dry ;  after  which,  in  the  case  of 
cover-glass  preparations,  they  are  inverted  upon  a  drop  of 
( 1anada  balsam  on  a  slide  and  examined  with  the  oil-immer- 
sion lens ;  or,  when  slides  have  been  prepared,  after  wash- 


44        EXAMINATION  AND  STAINING   OF  BACTERIA. 

ing  and  drying  a  drop  of  cedar  oil  is  put  over  the  prepa- 
ration, and  the  same  is  examined  with  the  oil-immersion 
objective  without  the  use  of  a  cover-glass. 

Air-bubbles  are  often  caught  between  the  balsam  and  the 
cover-glass.  Gentle  uniform  pressure  begun  at  the  centre  of 
the  cover-glass  and  progressively  applied  toward  its  periphery 
will  ordinarily  remove  them.  If  this  should  fail,  heat  care- 
fully applied  until  the  balsam  is  quite  soft  will  aid  in  the 
riddance  of  the  others. 

IV.  The  Special  Methods  of  Staining. 

As  stated  above,  this  method  may  be  applied  for  the  stain- 
ing of  nearly  all  bacteria.  Some,  however,  are  not  so  easily 
stained,  and  special  methods  must  be  resorted  to  to  increase 
the  penetrating  power  of  the  dye.  The  most  commonly  used 
will  be  here  described. 

1.  Loeffler's  Method. 

In  this  method,  instead  of  using  the  ordinary  watery  solu- 
tion of  an  anilin  dye,  Loeffler's  alkaline  solution  of  methylene- 
blue  is  used.  This  is  prepared  as  follows  : 

Concentrated  alcoholic  solution 

of  methylene-blue,  30 parts; 

Caustic  potash  solution 

(1:10,000),  100     "    . 

Mix  well  and  filter. 

This  method  stains  well  all  the  ordinary  bacteria,  but  is 
specially  useful  for  the  staining  of  the  bacillus  of  diphtheria. 

2.  Koch-Ehrlich's  Method. 

Anilin  water  is  prepared  by  adding  a  few  drops  of  anilin 
oil,  drop  by  drop,  to  distilled  water  in  a  test-tube,  shaking 
well  after  the  addition  of  each  drop  and  until  the  liquid 
assumes  a  milky  appearance,  after  which  it  is  filtered  through 
moistened  filter-paper  until  the  filtrate  is  absolutely  clear. 

To  100  parts  of  this  clear  filtrate  of  anilin  water,  10  parts 


THE  STAINING   OF  BACTERIA.  45 

of  absolute  alcohol  and  10  parts  of  an  alcoholic  solution  of 
fuchsin,  methylene-blue,  or  gentian-violet,  are  added,  and  the 
whole  thoroughly  mixed  and  filtered.  The  preparation  is 
better  when  made  fresh  in  small  quantity  at  each  time  it  is 
needed,  as  it  decomposes  in  a  few  days. 

In  Koch-Ehrlich's  method  this  anilin  water  (violet,  blue, 
or  red)  is  used  instead  of  the  simple  watery  solution  of  the 
dye.  It  possesses  much  more  penetrating  power,  and  this 
again  may  be  increased  by  gently  heating  the  slide  or  cover- 
glass,  over  a  Bunsen  burner,  while  it  is  being  stained. 

It  is  applicable  whenever  it  is  desirable  so  to  fix  the  color 
in  the  bacteria  that  one  may  by  means  of  decolorizing  agents 
remove  the  color  from  the  surrounding  objects  and  tissue  and 
fix  it  solely  on  the  bacteria  themselves.  Some  bacteria  so 
retain  the  color  by  this  method  that  it  serves  to  distinguish 
them  from  others  which  they  very  much  resemble,  but  which 
do  not  possess  the  same  persistent  retention  of  the  stain. 

The  usual  decolorizing  agent  employed  is  either  ordinary 
alcohol  or  diluted  sulphuric  or  hydrochloric  acid  (1  : 4). 

3.  Gram's  Method. 

Treat  the  object  to  be  colored  with  anilin-water  gentian- 
violet  for  about  three  minutes,  after  which  immerse  in  Gram's 
fluid.  This  consists  of: 

Iodine,  1  part; 

Iodide  of  potassium,  2  parts  ; 

Water,  300      "    . 

Maintain  this  immersion  for  five  minutes,  then  pass  the 
preparation  through  alcohol  and  rinse  in  water.  If  the 
object  is  still  of  a  violet  color,  treat  it  again  with  Gram's 
fluid  and  alcohol  until  no  violet  is  visible  to  the  naked  eye. 
This  method  is  applicable  to  a  number  of  bacteria,  and  serves 
as  a  mode  of  differentiation  between  some  of  them  which 
could  not  otherwise  be  distinguished  under  the  microscope. 
It  serves  also  to  color  the  capsule  of  bacteria,  and,  slightly 
modified,  is  useful  for  the  stain  of  bacteria  in  tissue.  A 
contrast-color  should  be  given  to  the  uncolored  parts. 


46        EXAMINATION  AND  STAINING   OF  BACTERIA. 

4.  Ziehl's  Carbol-fuchsin  Method. 
Make  a  solution  of  carbol-fuchsin  as  follows  : 

Fuchsin,  1  part; 

Crystallized  carbolic  acid,  5  parts ; 

Alcohol,  10      "    ; 

Water,  100      "    . 

Immerse  the  glass  in  or  cover  same  with  the  carbol-fuchsin 
solution,  heat  gently  over  the  flame  of  a  Bunsen  burner,  gradu- 
ally bringing  to  a  point  just  below  boiling;  repeat  this  two 
or  three  times;  after  which  immerse  in  nitric  acid  solution 
(1  part  of  acid  to  3  parts  of  water)  until  the  color  is  scarcely 
visible  to  the  naked  eye.  To  ascertain  this,  wash  off  the  acid 
from  the  film  with  water.  If  color  is  still  faintly  visible, 
remove  it  by  dipping  into  alcohol ;  wash  in  water,  dry, 
mount  in  Canada  balsam,  and  examine.  A  contrast-color 
may  be  given  to  the  rest  of  the  specimen  by  employing 
methylene-blue. 

This  is  a  useful  method  in  coloring  cover-glass  preparations 
for  tubercle  bacilli  or  for  the  Bacillus  leprse. 

5.  Gabbett's  Method. 

IThis  is  a  modification  of  Ziehl's  method,  and  is  perhaps  the 
best  method,  on  account  of  its  simplicity  and  rapidity,  for  the 
staining  of  the  tubercle  bacillus  in  secretions.  It  is  as  fol- 
lows :  Prepare  a  slide  or  cover-glass  film  as  indicated,  im- 
merse in  Ziehl's  carbol-fuchsin  solution  for  ten  minutes,  remove 
to  Gabbett's  sulphuric  acid  methylene-blue  solution  for  three 
to  five  minutes,  rinse  in  water,  dry,  mount,  examine.  The 
tubercle  bacilli  are  colored  red  and  the  other  bacteria  and  cell- 
nuclei  are  colored  blue. 

Gabbett's  solution  consists  of : 

Methylene-blue,  1  to  2  parts  ; 

Sulphuric  acid,  25  "     ; 

Water,  75  "     . 


THE  STAINING   OF  BACTERIA.  47 

Besides  the  foregoing  means,  which  will  stain  the  bacteria 
in  films,  bacteriologists  adopt  special  methods  for  the  staining 
of  bacteria  in  tissues,  and  for  the  staining  of  spores,  flagella, 
and  the  capsules  of  bacteria. 

V.  The  Staining  of  Capsules.  VJx 

1.  Welch's  Glacial  Acetic  Acid  Method. 

Prepare  the  cover-glass  in  the  usual  way.  Cover  the  film 
with  glacial  acetic  acid,  pour  off  the  acid  immediately,  do  not 
wash  in  water,  cover  the  film  with  anilin-water  gentian- violet 
solution  for  three  or  four  minutes,  wash  in  a  0.5  to  2  percent, 
solution  of  sodium  chloride,  dry,  mount,  and  examine. 

The  acetic  acid  coagulates  the  mucin  of  the  capsules  and 
renders  the  same  distinctly  visible. 

2.  Johne's  Method. 

A  cover-glass  film  prepared  in  the  usual  way  is  covered 
with  a  solution  of  gentian-violet  and  heated  until  steam  rises. 
The  stain  is  then  washed  oif  in  water,  and  the  cover-glass  put 
into  a  2  per  cent,  acetic  acid  solution  for  from  ten  to  fifteen 
seconds.  It  is  again  washed  in  water,  dried,  and  mounted  in 
balsam. 

VI.  The   Staining  of  Spores. 

Though  with  the  ordinary  method  of  staining,  spores  in 
bacteria  may  be  recognized  by  their  highly  refractive  appear- 
ance and  by  the  fact  that  they  have  not  taken  the  color,  they 
may  be  stained  themselves,  however,  by  special  methods. 

The  First  Method  (Abbott's). 

A  cover-glass  preparation  is  covered  with  Loeffler's  alkaline 
methylene-blue  solution  and  held  by  its  edge  with  forceps  over 
the  Bunsen  burner  flame  until  the  fluid  begins  to  boil.  It  is 
then  removed  from  the  flame,  and  after  a  few  seconds  heated 
again.  This  step  is  repeated  a  number  of  times  for  one  or 
two  minutes,  after  which  it  is  washed  in  water,  and  decolor- 


48     EXAMINATION  AND   THE  STAINING   OF  BACTERIA. 

ized,  until  all  blue  coloring  visible  to  the  naked  eye  has  dis- 
appeared, in  the  following  solution  : 

Alcohol  (80  per  cent.),  98  parts ; 

Nitric  acid,  2      "     . 

The  cover-glass  is  then  dipped  for  a  few  seconds  into  the  fol- 
lowing solution  : 

Saturated  alcoholic  solution  of  eosin,    10  parts  ; 
Water,  90      "     . 

After  this  it  is  again  rinsed  in  water,  dried,  and  mounted. 

The  Second  Method,     "if  tfV  ^^ 

Float  a  cover-glass  preparation  film-side  down  in  a  watch- 
glass  full  of  Koch-Ehrlich's  fuchsin  solution.  Take  the  watch- 
glass  by  its  edge  with  a  pair  of  forceps  and  hold  same  over 
a  low  Bunsen  flame  until  the  staining  fluid  begins  to  boil. 
Remove  from  the  burner,  and  after  a  few  minutes  repeat  this 
process  five  or  six  times.  After  cooling,  the  cover-glass, 
without  washing  in  water,  is  transferred  to  a  second  watch- 
glass  containing  a  decolorizing  solution  as  follows  : 

Absolute  alcohol,  100  parts  ; 

Hydrochloric  acid,  3      "     . 

Place  the  cover-glass,  film-side  up,  at  the  bottom  of  this 
watch-glass  and  let  it  remain  for  one  or  two  minutes. 
Remove,  wash  in  water,  stain  with  methylene-blue  solution 
for  one  or  two  minutes,  wash  rapidly  in  water,  dry,  and 
mount. 

By  this  method  the  spores  will  be  stained  red  and  the  body  of 
the  bacteria  cells  blue. 

The  Third  Method. 

Cover-glasses  are  prepared  in  the  usual  way.  After  fix- 
ing, the  preparation  is  immersed  in  chloroform  for  two  min- 
utes, washed  in  water,  placed  for  one  or  two  minutes  in  a  5 


THE  STAINING   OF  BACTERIA.  49 

per  cent,  solution  of  chromic  acid,  again  washed  in  water,  and 
stained  in  hot  Ziehl's  carbol-fuchsin  solution  for  five  minutes. 
The  staining  fluid  is  poured  off  and,  without  washing  in  water, 
the  preparation  is  decolorized  in  5  per  cent,  sulphuric  acid. 
After  this  it  is  again  washed  in  water,  and  finally  stained  for 
two  or  three  minutes  in  the  watery  methylene-blue  solution. 

The  spores  will  be  stained  red,  the  body  of  the  cells  blue. 

The  action  of  the  chloroform  is  to  dissolve  the  fatty  crys- 
tals that  may  be  in  the  preparation.  The  chromic  acid  acts 
on  the  membrane  of  the  spores  and  permits  the  entrance  of 
the  stain. 

The  Fourth  Method  (Fiocca's). 

Ten  to  20  parts  of  an  alkaline  solution  of  an  anilin  color 
are  added  to  20  c.c.  of  a  10  per  cent,  ammonia  solution  in  a 
watch-glass  ;  then  heat  is  applied  until  steam  commences  to 
be  given  off.  The  cover-glass  stays  in  this  hot  solution  from 
three  to  five  minutes  ;  it  is  then  taken  out  and  washed  in  a 
20  per  cent,  solution  of  nitric  or  sulphuric  acid  to  decolorize  it, 
then  washed  again,  when  a  contrast-color  may  be  given. 

It  must  be  remembered  that  there  is  considerable  difference  in 
the  behavior  of  spores  of  different  bacteria  to  the  staining  methods 
described;  some  stain  very  readily  and  others  with  considerable 
difficulty. 

Practice  will  be  the  best  guide  as  to  which  method  is  best  to 
employ  and  to  what  extent  it  should  be  carried  out.  ^y 

VII.  The  Staining  of  Flagella. 

The  hair-like  processes  of  the  bacteria  which  serve  for 
their  locomotion,  the  flagella,  can  not,  on  account  of  their 
fineness,  be  seen  in  any  stained  specimen,  nor  can  they  be 
stained  by  any  of  the  'ordinary  methods  just  described  for 
staining  bacteria.  In  order  to  make  them  visible,  it  is  neces- 
sary to  use  special  stains  in  which  the  action  of  mordants  plays 
an  essential  part. 

1.  Loeffler's  Method. 

This  is  the  most  common  method,  arid  is  as  follows :  Clean 
ve'ry  carefully  a  thin  cover-glass,  and  spread  very  thinly  and 

4— M.  B. 


50        EXAMINATION  AND  STAINING   OF  BACTERIA. 

evenly  upon  it  as  few  as  possible  of  the  bacteria  to  be  exam- 
ined. This  is  done  by  diluting  with  sterilized  water  a  number 
of  times  the  culture  containing  them.  The  cover-glass  is  dried 
and  fixed  in  the  ordinary  way.  The  following  solution, 
known  as  a  mordant,  is  then  applied  : 

Tannic  acid  (20  per  cent,  solution  in 

water,  filtered),  10  parts; 

Cold    saturated    solution    of    ferrous 

sulphate,  filtered,  5      "    ; 

.  y  Saturated  alcoholic  solution  of  fuchsin,   1  part. 

+  Ho  7«.a  Jrti    OK  tM.#&>  v 

A  few  drops  of  it  are  placea  on  the  film,  and  the  cover-glass 
taken  up  with  a  pair  of  forceps  and  held  over  the  flame  of  a 
Bunsen  burner  until  the  solution  begins  to  steam,  but  not 
allowing  the  boiling-point  to  be  reached.  It  is  next  washed 
rapidly  in  water,  and  then  in  absolute  alcohol.  The  bacteria 
are  to  be  stained  in  anilin-water  fuchsin  solution  in  the  ordi- 
nary way. 

Practice  has  shown,  however,  that  different  bacteria  behave 
differently  when  exposed  to  this  staining,  and  Loeffler  himself 
has  modified  it  to  meet  these  requirements.  Having  found 
that  the  addition  of  an  alkali  favors  the  staining  of  flagella 
in  some  of  the  bacteria,  he  has  added  to  his  stain  1  per  cent. 
of  sodium  hydrate.  In  other  cases,  having  found  that  an  acid 
helps  to  bring  out  the  flagella,  he  has  added  to  his  stain  a 
solution  of  sulphuric  acid  in  water  of  such  strength  that  1  c.c. 
will  neutralize  1  c.c.  of  the  sodium  hydrate  solution. 

The  following  bacteria  require  an  acid  solution  added  to  the 
stain  :  Bacillus  pyocyaneus,  the  spirillum  of  Asiatic  cholera, 
Spirillum  rubrum,  Spirillum  concentricum,  Spirillum  Metch- 
nikowi. 

The  following  bacteria  require  an  alkaline  addition  to  the 
staining  solution :  Bacillus  mesentericus,  Micrococcus  agilis, 
Bacillus  typhosusj  Bacillus  subtilis,  bacillus  of  malignant 
oedema,  Bacillus  anthracis  symptomatici. 

In  a  general  way  one  may  say  that  bacteria  that  produce  acid 
in  the  media  in  which  they  grow  require  the  addition  of  an  alkali 


THE  STAINING   OF  BACTERIA.  51 

to  the  mordant,  and  those  which  produce  alkalies  require  the 
addition  of  an  acid. 

2.  Bunge's  Method. 

Bunge's  method,  a  modification  of  Loeffler's  method,  is  as 
follows  :  Prepare  a  saturated  solution  of  tannin  in  water,  and 
also  a  5  per  cent,  solution  of  sesquichloride  of  iron  in  distilled 
water;  to  3  parts  of  the  tannin  solution  add  1  part  of  the 
iron  solution.  To  10  parts  of  such  a  mixture  add  1  part  of 
concentrated  watery  solution  of  fuchsin.  This  mordant  should 
never  be  used  j resit ,  but  only  after  it  has  been  exposed  to  the  air 
for  several  days.  The  cover-glass,  thoroughly  cleaned,  is 
covered  over  by  this  mordant  for  five  minutes,  after  which 
it  is  slightly  warmed.  It  is  then  washed,  dried,  and  stained 
faintly  with  a  little  carbol-fuchsin. 

3.  Pitfield's  Method. 

The  following  solution  is  used  as  a  mordant : 

Tannic  acid  (10  per  cent,  solution, 

filtered),  10  parts; 

Corrosive  sublimate  (saturated  aque- 
ous solution),  5  "  ; 

Alum  (saturated  aqueous  solution),  5     "     ; 

Carbol-fuchsiri,  5     "     . 

Let  this  stand,  and  pour  off  the  clear  fluid.     This  mordant 
will  keep  for  one  or  two  weeks. 

The  staining  fluid  is  prepared  as  follows  : 

Alum  (saturated  aqueous  solution),  10  parts; 
Gentian-violet   (saturated    alcoholic 

solution),  2      "    . 

This  stain  is  to  be  prepared  fresh  every  second  or  third  day. 

The  modus  operandi  is  as  follows :  On  an  absolutely  clean 
cover-glass  make  a  thin  film  as  already  described.  Treat  the 
film  with  the  mordant  applied  cold  for  twenty-four  hours,  or 


52         EXAMINATION  AND  STAINING   OF  BACTERIA. 

with  the  mordant  applied  steaming,  but  not  boiling,  for  three 
minutes ;  wash  off  thoroughly  in  water  and  dry ;  treat  with 
the  stain  in  the  same  manner  as  with  the  mordant ;  wash  in 
water,  dry,  mount,  and  examine  with  an  oil-immersion  lens. 
Other  methods,  such  as  Van  Ermengem's,  BowhilTs,  etc.,  are 
highly  recommended,  but  in  the  author's  hands  have  given 
no  better  result  than  the  foregoing  three  methods. 

VIII.  The  Staining  of  Bacteria  in  Tissues. 
1.  The  Staining  of  Sections. 

Sections  should  be  cut  in  the  ordinary  way  in  paraffin  or 
celloidin.  The  sections  are  first  put  into  water  for  a  few 
minutes,  then  transferred  to  watch-glasses  containing  watery 
solutions  of  any  basic  anilin  dye,  and  allowed  to  remain  from 
five  to  ten  minutes ;  they  are  next  removed,  rinsed  in  water, 
decolorized  in  a  0.1  solution  of  acetic  acid  for  a  few  seconds, 
again  washed  in  water,  then  for  a  few  minutes  in  absolute 
alcohol,  and  placed  in  cedar  oil  or  xylol.  They  are  allowed 
to  remain  in  xylol  from  one-half  to  one  minute.  They  are 
finally  spread  thinly  on  a  spatula  and  brought  to  the  slides, 
where  the  excess  of  fluid  is  taken  up  with  blotting-paper ; 
after  which  a  drop  of  xylol  balsam  is  placed  on  the  sections, 
which  are  covered  by  thin  clean  cover-glasses,  when  they  are 
ready  for  examination. 

2.  Gram's  Method  for  Staining  Bacteria  in  Tissue. 

This  is  practically  the  same  as  the  method  for  cover-glass 
preparations. 

The  section  is  stained  in  anilin-water  gentian-violet  (Koch- 
Ehrlich)  diluted  with  one-third  its  volume  of  water.  The 
section  remains  in  this  for  about  ten  minutes  at  the  tem- 
perature of  the  incubator.  From  this  it  is  taken  out  and 
washed  alternately  in  Gram's  iodine  solution  and  alcohol  until 
all  the  naked-eye  color  has  been  extracted.  It  is  then  put 
into  a  watery  solution  of  eosin  or  Bismarck-brown  for  one 
minute,  again  washed  in  alcohol  a  few  seconds,  and  then  put 


THE  STAINING   OF  BACTERIA.  53 

for  one-quarter  minute  in  absolute  alcohol.  After  this  it  is 
transferred  to  xylol  for  one-half  minute,  then  lifted  to  a  slide, 
mounted  in  Canada  balsam,  and  examined. 

3.  Weigert's  Modification  of  Gram's  Method. 

Stain  sections  in  the  Koch-Ehrlich  anilin- water  gentian-violet 
solution  for  five  or  ten  minutes  ;  wash  them  afterward  in 
water  or  physiological  salt  solution.  Transfer  to  slide  and 
remove  excess  of  fluid  with  blotting-paper.  Treat  with  the 
iodine  solution  of  Gram  for  three  minutes.  Take  up  the  ex- 
cess of  solution  with  blotting-paper.  Cover  the  section  with 
anilin  oil,  wash  out  the  oil  with  xylol,  and  mount  in  xylol 
balsam.  The  anilin  oil  in  this  case  acts  as  a  decolorizing 
agent,  and  should  be  removed  carefully,  otherwise  the  speci- 
men will  not  keep. 

4.  Kuehne's  Carbolic  Methylene-Blue  Method. 

Put  the  sections  into  the  following  solution  for  one-half 
hour : 

Methylene-blue  in  substance,  1^  part ; 

Absolute  alcohol,  10    parts. 

Rub  thoroughly  in  a  mortar,  and  when  the  blue  is  completely 
dissolved  add  100  parts  of  a  5  per  cent,  carbolic  acid  solu- 
tion. This  solution  should  be  made  fresh  when  needed. 
The  sections  are  stained  for  fifteen  minutes  in  this  solution 
and  then  washed  in  water  until  free  from  it.  They  are 
next  transferred  to  a  2  per  cent,  hydrochloric  acid  solution, 
then  to  a  solution  of  carbonate  of  lithium  (of  the  strength  of 
6  to  8  drops  of  a  concentrated  watery  solution  of  the  salt 
to  10  drops  of  water),  and  from  this  they  are  again  washed 
in  water  and  in  absolute  alcohol  containing  sufficient  meth- 
ylene-blue  in  substance  to  give  it  a  blue  color,  then  for  a  few 
minutes  in  anilin  oil  to  which  a  little  methylene-blue  in  sub- 
stance has  been  added,  and  they  are  then  rinsed  out  in  pure 
anilin  oil ;  from  this  they  are  placed  in  oil  of  turpentine  or 
thymol  for  two  minutes,  then  in  xylol,  and  mounted  in  xylol 
balsam.  j  ^  foJ^  ^'iM^rwvi  Qu^JU^* 

*' 

,. 


54         EXAMINATION  AND  STAINING   OF  BACTERIA. 

The  advantages  of  this  method  are  that  it  is  generally  ap- 
plicable. Bacteria  are  not  robbed  of  their  color,  and  the 
tissue  is  sufficiently  decolorized  to  render  the  bacteria  visible 
and  to  admit  of  a  contrast-stain. 

5.  Ziehl-Neelsen's  Method. 

The  sections  are  warmed  in  a  solution  of  carbol-fuchsin  for 
one  hour  at  a  temperature  of  about  45°  to  50°  C.,  decolorized 
for  a  few  seconds  in  a  5  per  cent,  sulphuric  acid  solution,  then 
put  into  70  per  cent,  alcohol,  then  in  absolute  alcohol  for  a  few 
seconds  to  dehydrate,  then  in  xylol  to  clear,  and  mounted 
on  a  slide  in  xylol  balsam. 

QUESTIONS. 

What  powers  of  the  microscope  are  necessary  for  the  examination  of 
bacteria  ? 

How  are  bacteria  examined  alive  ? 

How  is  a  hanging-drop  preparation  made  ? 

What  is  the  usual  method  of  staining  bacteria? 

What  are  the  most  usual  stains  used  for  bacteria? 

What  is  Loeffler's  method  ? 

Describe  the  Koch-Ehrlich  method. 

What  are  the  usual  decolorizing  agents  used  ? 

What  is  Gram's  method  ? 

Describe  Ziehl's  carbol-fuchsin  method. 

Describe  Gabbett's  method. 

Give  Welch's  method  of  staining  the  capsule  of  bacteria. 

Give  Johne's  method. 

Give  Abbott's  method  of  staining  spores.  Moeller's  method.  Fiocca's 
method. 

Give  Loeffler's  method  of  staining  flagella. 

Which  bacteria  require  the  addition  of  acid  to  the  mordant  in  order  to 
stain  their  flagella?  Which  require  the  addition  of  alkalies? 

Describe  Bunge's  method  of  staining  flagella.     Pitfield's  method. 

How  would  you  stain  bacteria  in  tissue  ?  Give  Gram's  method.  Weigert's 
method.  Kuhne's  method.  Ziehl-Neelsen's  method. 


THE  CULTIVATION  OF  BACTERIA.  55 


CHAPTER   III. 

THE  PROCESS,  MEDIA,  AND   UTENSILS  OF  THE  CULTI- 
VATION  OF  BACTERIA. 

THE  PROCESS  OF  THE  CULTIVATION  OF  BACTERIA. 

As  mentioned  before,  bacteria  can  not  be  separated  from 
one  another  by  form  and  appearance  under  the  microscope 
only.  Indeed,  in  a  number  of  instances,  and  even  with  the 
highest  power  of  the  microscope,  some  very  inoffensive  bac- 
teria resemble  very  much  and  can  not  be  differentiated  from 
some  that  are  highly  pathogenic.  Especially  is  this  the  case 
with  the  group  of  cocci. 

In  all  such  cases  it  is  necessary  to  study  the  properties 
and  mode  of  growth,  and  for  this  purpose  the  bacteriologist 
must  use  and  prepare  suitable  soils,  which  are  known  by  the 
name  of  culture -media.  These  culture-media  must  themselves 
be  absolutely  free  from  all  live  bacteria — that  is,  sterile ;  or, 
if  they  naturally  contain  bacteria,  or  if  bacteria  have  been 
introduced  during  their  preparation,  these  must  be  destroyed, 
or,  in  bacteriological  language,  the  media  must  be  sterilized. 

THE  MEDIA  OF  THE  CULTIVATION  OF  BACTERIA. 

All  substances  that  contain  carbon  and  nitrogen  compounds  in 
assimilable  form  associated  with  water  may  be  used  as  culture- 
soils  for  bacteria,  The  culture-media  used  ordinarily  are 
either  natural  or  artificial.  They  may  be  liquid  or  solid ;  or, 
again,  they  may  be  solid  at  the  temperature  used,  and  liquefied 
at  a  temperature  not  high  enough  to  destroy  bacterial  life. 

I.  The  Most  Commonly  Used  Liquid  Culture -Media. 

1.  Milk. 

Milk,  as  contained  in  the  udders  of  the  cow,  is  an  excel- 
lent culture-medium,  and  is  generally  sterile.  In  its  col- 
lection, however,  it  usually  becomes  contaminated — that  is, 
bacteria  are  introduced  into  the  milk  :  so  much  so  that  it  is 


56  THE  CULTIVATION  OF  BACTERIA. 

necessary  to  sterilize  the  same  before  using,  and  for  this  pur- 
pose what  is  known  as  the  discontinuous  or  fractional  steril- 
ization by  steam  is  resorted  to. 

Mode  of  Preparing  Sterilized  Milk. — Sterilized  test-tubes 
from  5  to  7  inches  in  length,  and  about  from  1  to  1J  inches 
in  diameter,  are  filled  to  one-third  their  capacity  with  raw 
milk.  The  test-tubes  are  plugged  tightly  with  ordinary 
cotton-batting,  and  are  submitted  to  live  steam  in  the  steam 
sterilizer,  at  100°  C.,  for  twenty  minutes  each  time,  on  three 
consecutive  days. 

Before  sterilization  tincture  of  blue  litmus  may  be  added  to 
the  milk,  and  in  this  way  the  generation  of  acids  by  bacteria 
may  easily  be  ascertained. 

Milk  prepared  in  the  foregoing  manner  offers  an  excellent 
culture-soil  for  nearly  all  forms  of  bacteria ;  it  serves  also  for 
differentiating  between  certain  species  accordingly  as  these 
have  the  property  of  coagulating  the  casein  in  the  milk 
rapidly,  slowly,  or  not  at  all. 

2.  Animal  Blood-Serum. 

Animal  blood-serum  obtained  from  a  slaughter-house  is  an 
exceedingly  useful  culture-medium. 

Its  mode  of  preparation  is  as  follows  :  In  large  cylindrical 
jars  the  fresh  fluid  blood  is  collected  and  allowed  to  remain 
untouched  for  a  half-hour  or  an  hour.  After  this,  with  a 
clean  sterilized  glass  rod  the  coagulum  that  begins  to  form  is 
detached  from  the  sides  of  the  vessel.  The  vessel  then,  well 
covered  and  protected  from  dust,  is  put  into  an  ice-box,  and 
at  the  end  of  twenty-four  hours  the  clot,  consisting  of  fibrin 
and  of  blood-corpuscles,  is  firm  and  sinks  to  the  bottom  of 
the  vessel,  leaving  a  clear  serum  above  and  around  it.  This 
clear  serum  may  be  siphoned  or  pipetted  out  and  distributed 
among  sterilized  test-tubes,  which,  after  being  plugged  with 
absorbent  cotton-batting,  are  sterilized  in  Koch's  serum  steril- 
izer by  the  low  temperature  process  to  be  described  later. 

Loeffler's  modification  of  this  method  is  generally  used  in  all 
municipal  laboratories  for  the  cultivation  and  diagnosis  of 
the  bacillus  of  diphtheria  ; 


THE  MEDIA   OF  THE  CULTIVATION  OF  BACTERIA.     57 

Beef  or  mutton-blood  is  collected  in  the  usual  way,  and  to 
8  parts  of  the  clear  serum  1  part  of  glucose-bouillon  is  added. 
This  mixture  distributed  among  test-tubes  is  sterilized  and 
hardened  in  a  slanting  position  in  a  steam  sterilizer  at  a  tem- 
perature between  80°  and  90°  C.,  for  an  hour  each  day  dur- 
ing a  whole  week. 

3.  The  serum  of  ascitic  fluid  and  (4)  the  fluid  of  hydrocele 
are  sometimes  used  for  the  cultivation  of  bacteria,  and  are 
prepared  in  the  same  manner  as  ordinary  blood-serum. 

5.  Urine. 

Urine  may  also  be  used  for  the  cultivation  of  bacteria. 
For  this  purpose  it  is  obtained  by  means  of  a  sterilized  cathe- 
ter directly  from  the  bladder,  where  it  is  generally  sterile. 
It  is  safest,  however,  to  sterilize  it  by  steam  for  one  hour 
before  use. 

6.  Pasteur's  Solution. 

Filtered  water,  100  parts ; 

Cane-sugar,  10     "     ; 

Ammonium  tartrate,  1  part. 

With  the  addition  of  1  part  of  the  ashes  of  yeast  this  was 
formerly  extensively  used  as  a  culture-medium,  but  is  now 
seldom  used. 

7.  Bouillon. 

Bouillon  is  the  most  frequently  used  of  all  the  fluid  media. 
It  is  prepared  as  follows  :  1  pound  of  fresh  lean  beef  is 
chopped  up  very  fine  and  covered  with  1  liter  of  sterilized 
water,  and  put  into  an  ice-box  for  twenty-four  hours,  after 
which  the  aqueous  extract  is  obtained  by  filtration  through 
muslin  by  pressure,  sufficient  water  being  added  if  necessary 
to  make  up  the  original  liter.  To  this  filtrate  10  grams  of 
peptone  and  5  grams  of  sodium  chloride  are  added,  and  the 
whole  is  cooked  on  a  water-bath  or  in  an  enamelled  iron 
kettle  for  a  half-hour,  after  which  sufficient  of  a  saturated 
solution  of  sodium  carbonate  is  added  drop  by  drop  to  give 
the  mixture  a  slight  alkaline  reaction.  This,  after  cooking 


58  THE  CULTIVATION  OF  BACTERIA. 

for  fifteen  or  twenty  minutes,  is  filtered  through  absorbent 
cotton  several  times  into  test-tubes  and  sterilized  by  steam 
for  twenty  minutes  on  three  successive  days. 

The  addition  of  5  per  cent,  neutral  glycerin  to  this  bouillon 
makes  an  excellent  liquid  culture-medium  for  tubercle  bacilli, 
and  is  highly  recommended  by  Roux. 

Any  standard  beef-extract,  such  as  Liebig's,  Armour's, 
Wyeth's,  etc.,  may  be  used  in  making  this  bouillon,  instead 
of  the  meat  itself,  5  grams  of  the  extract  being  added  to 
1  liter  of  water,  the  rest  of  the  process  being  the  same. 

pjCL^DK^X  \  Q<fat  V-twt  A*J**  -  IL^tO?  teOJ&aSL*^^ 

II.  The  Most  Commonly  Used  S6lid  Culture-Media.fU^ 

1.  Gelatin. 

Nutrient  gelatin  is  prepared  as  follows  :  A  meat-infusion, 
as  described  for  bouillon,  is  prepared  and  put  into  a  large 
Bohemian  flask  of  2  liters  capacity ;  to  this  meat-infusion  are 
added  : 

Chloride  of  sodium,  5  grams  ; 

Peptone,  10      "      ; 

Best  quality  gelatin  100      " 

The  flask,  loosely  closed  with  a  plug  of  absorbent  cotton,  is 
cooked  over  a  water-bath  until  all  the  gelatin  is  dissolved. 
This  will  take  from  an  hour  and  a  half  to  two  hours.  After 
this  the  mixture,  which  will  be  found  to  be  decidedly  acid, 
is  neutralized  by  means  of  a  solution  of  sodium  carbonate 
added  drop  by  drop  until  the  mixture  is  faintly  alkaline,  as 
in  the  bouillon.  The  flask  is  again  put  over  the  water-bath 
for  an  hour,  after  which  the  mixture  i-s  filtered  hot  through 
absorbent  cotton  and  sterilized  in  the  steam  sterilizer  for 
twenty  minutes  each  day  on  three  consecutive  days. 

Great  care  must  be  exercised  to  introduce  the  medium  into  the 
sterilizer  only  when  steam  is  actively  being  generated,  and  not  to 
allow  it  to  cool  in  the  sterilizer. 

Advantages. — Gelatin  is  a  most  excellent  medium,  and 
remains  solid  at  room  temperature  (22°  or  24°  C.),  but  is 
readily  liquefied  when  exposed  to  a  higher  temperature. 


THE  MEDIA    OF  THE  CULTIVATION  OF  BACTERIA.     59 

Not  only  does  it  serve  as  an  excellent  medium  for  the  culti- 
vation of  nearly  all  bacteria  that  grow  out  of  the  incubator 
temperature  (36°  to  37°  C.),  but  it  offers  by  the  plate-method 
one  of  the  best  culture-soils  for  isolating  bacteria. 

As  some  of  the  bacteria  liquefy  gelatin  and  others  do  not, 
it  is  useful  also  in  the  differentiation  of  these  species.  On 
account  of  its  clearness,  its  easy  preparation,  and  the  other 
advantages  mentioned  above,  it  is  one  of  the  best  culture- 
soils  available. 

The  disadvantages  lie  chiefly  in  the  fact  that  it  liquefies  at 
a  much  lower  temperature  than  that  of  the  incubator,  and 
can  not  therefore  be  used  for  the  cultivation  of  some  patho- 
genic bacteria  which  grow  only  at  blood  temperature. 

2.  Agar. 

Nutrient  agar  is  prepared  much  in  the  same  manner  as 
gelatin,  except  that  20  or  30  grams  of  2  or  3  per  cent,  agar 
are  added,  instead  of  the  gelatin,  to  the  meat-infusion,  and 
the  process  of  cooking  must  be  much  more  prolonged  (four 
to  five  hours),  as  the  agar  is  much  slower  in  dissolving.  It 
is  necessary  also  sometimes,  after  dissolving  the  agar  and 
neutralizing  the  mixture,  to  add  the  white  of  one  or  two  eggs 
in  order  to  clarify  the  solution  before  filtration.  This  is  done 
by  removing  the  flask  from  the  fire  and  allowing  it  to  cool  to 
a  temperature  below  70°  C.  After  clarification  the  mixture 
is  again  cooked  for  one  and  one-half  to  two  hours  and  filtered 
through  absorbent  cotton  two  or  three  times.  This  filtration 
is  a  much  more  difficult  process  than  with  gelatin,  and  must 
be  carried  on  in  the  steam  sterilizer.  After  filtration  the 
agar  is  distributed  among  test-tubes  for  use,  and  is  sterilized 
by  the  same  procedure  as  nutrient  gelatin. 

Nutrient  agar  has  a  melting-point  much  higher  than  that 
of  gelatin,  about  42°  C.,  so  that  it  may  be  used  for  the  culti- 
vation of  those  bacteria  which  grow  only  at  or  best  at  the 
temperature  of  the  human  body.  It  possesses  all  the  advan- 
tages of  gelatin,  but  is  not  so  clear  and  transparent,  and  is  not 
liquefied  by  the  secretions  of  any  known  bacteria.  It  is  useful 
also  for  the  isolation  of  bacteria  by  means  of  plate  cultures. 


60  THE  CULTIVATION  OF  BACTERIA. 

The  disadvantages  of  agar,  when  compared  with  gelatin, 
lie  chiefly  in  the  greater  difficulty  of  its  preparation,  and 
especially  of  its  nitration. 

3.  The  glycerin-agar  culture  is  obtained  by  adding  5  per 
cent,  of  neutral  glycerin  to  nutrient  agar  before  sterilization. 
This  makes  a  very  favorable   medium  for  the  cultivation  of 
the  tubercle  bacillus  and  of  some  other  pathogenic  bacteria. 

4.  A  mixture  of  agar  and  gelatin  in  bouillon  is  sometimes 
used  so  as  to  obtain  the  advantages  of  the  two  substances  com- 
bined.    This  mixture  is  prepared  from  bouillon  in  the  usual 
way  by  dissolving  in  the  bouillon  0.75  per  cent,  of  agar  and 
5  per  cent,  of  gelatin  in  the  manner  outlined  in  the  foregoing 
paragraphs. 

There  are  a  number  of  special  filtering  apparatus  constructed 
for  the  purpose  of  facilitating  the  filtration  of  agar.  None 
in  the  author's  estimation  has  any  advantages  over  filtration 
through  absorbent  cotton  in  the  steam  sterilizer. 

5.  Potato  Culture. 

Koch  called  attention  to  the  great  value  of  potato  cultures 
for  differentiating  species  of  bacteria. 

The  mode  of  preparing  potatoes  is  as  follows  :  A  sound 
potato,  of  good  size,  with  an  intact  epidermis,  is  chosen,  and 
thoroughly  washed  and  scrubbed  with  a  brush  to  remove  all 
dirt ;  after  which,  with  a  sharp-pointed  knife,  all  the  eyes 
and  discolored  parts  are  cut  out  of  the  potato.  It  is  then 
washed  again  in  water  and  put  into  a  solution  of  1 : 500 
bichloride  of  mercury  for  an  hour,  after  which  it  is  cooked  in 
the  steam  sterilizer  for  forty  minutes  on  each  of  two  suc- 
cessive days.  Just  before  inoculation  the  potato  is  split  into 
halves  by  means  of  a  sterilized  knife  and  allowed  to  fall  into 
a  moist  chamber  cut  surface  up. 

The  moist  chamber  consists  of  a  double  glass  dish,  the  upper 
one  of  which  is  larger  than  the  lower.  This  chamber  should 
be  thoroughly  rinsed  with  a  1  : 500  solution  of  bichloride  of 
mercury  before  use. 

Some  precautions  are  necessary  to  prevent  contamination 
of  the  potato  from  external  germs.  The  hand  that  cuts  the 


THE  MEDIA    OF  THE  CULTIVATION  OF  BACTERIA.     61 

potato  should  be  thoroughly  sterilized  in  a  1  : 1000  bichloride 
of  mercury  bath.     It  is  better  also  after  washing  the  potato 
and  before  submitting  it  to  the  bichloride  bath  to  wrap  it  in 
some  thin  tissue  paper,  and  to  keep  it  in  this  paper 
until  ready  for  inoculation.  FlG<  11- 

Preparation  of  a  Potato  for  Test-tube  Culture. — By 
means  of  a  cork-borer  (Fig.  10)  a  cylinder  is  cut 
from  a  sound  potato.  This  cylinder  is  cut  obliquely 

FIG.  10. 


Nest  of  cork-borers,  used  to  cut  potatoes  for  test-tube  cultures. 

into  two  pieces,  and  each  placed  into  a  large  test- 
tube  (Fig.  11),  in  which  it  is  sterilized  and  cooked 
on  three  successive  days  for  a  half-hour  in  a  steam 
sterilizer. 

6.  Potato-paste  is  sometimes  used  for  cultivation. 
For  this  purpose  a  potato  is  boiled,  peeled,  and 
mashed  with  a  little  sterilized  water,  placed  in  a 
suitable  glass  dish,  and  sterilized  for  one-half  hour 
on  three  successive  days  in  a  steam  sterilizer. 

7.  Bread-paste  is  a  useful  medium  for  the  growth 

of  moulds,  and  is  made  in  the  same  way  as  potato-  PotatSbe.test 
paste. 

III.  The  Most  Commonly  Used  Special  Culture-Media. 

In   making  final  distinctions  between   the  different  species 
of  bacteria  the  following  special  media  are  occasionally  used  : 

1.  The  Peptone  Solution. 

Dry  peptone,  1    part; 

Sodium  chloride,  J     "    ; 

Distilled  water,  100    parts. 

This  is  filtered,  decanted  into  test-tubes,  and  sterilized  in  the 
steam  sterilizer. 


62 


THE  CULTIVATION  OF  BACTERIA. 


This  preparation  is  chiefly  used  for  determining  whether  the 
bacteria  secrete  indol  or  not.  It  is  necessary  therefore  to  see 
that  the  peptone  preparation  used  be  chemically  pure,  also 
that  the  solution  be  free  from  the  presence  of  carbohydrates. 

2.  Glucose-,  lactose-,  and  saccharose-bouillon.  These  are  made 
by  adding  to  the  bouillon  after  filtration  and  before  steriliza- 
tion from  1  to  2  per  cent,  of  the  desired  kind  of  sugar. 

THE    UTENSILS   OP   THE   CULTIVATION   OF    BACTERIA. 

For  making  and  keeping  cultures  the  following  instruments, 
glassware,  and  utensils  are  required  : 

1.  Perfectly  clean  glass  tubes,  5  to  7  inches  long  and  J  to 

FIG.  12. 


FIG.  13. 


Glass  test-tube. 


Erlenmeyer  flask. 


1J  inches  in   diameter  (Fig.  12). 
with  ordinary  cotton-batting. 
2.  Erlenmeyer  flasks  (Fig.  13). 


These  should  be  plugged 


THE   UTENSILS  OF  THE  CULTIVATION  OF  BACTERIA.    63 

3.  Cylindrical  brushes  with  reed  handle,  and  wire-handled 
brush  of  Leutz  &  Sons,  for  the  purpose  of  cleaning  test-tubes 
(Fig.  14). 


FIG.  14. 


Brushes  for  cleaning  test-tubes :  a,  with  reed  handle  ;  6,  with  wire  handle. 

4.  Bohemian  glass  flasks  of  1  and  2  liters  capacity. 

5.  Platinum  needles,  straight  and  looped,  mounted  in  gL 
rods  (Fig.  15). 

FIG.  15. 


(a)  Looped  and  (6)  straight  platinum  wires  in  glass  handles. 

6.  Plates  of  ordinary  glass  about  4  by  5  inches,  and  Russia 
iron  boxes  to  hold  them  during  sterilization  (Fig.  16). 

FIG.  16. 


Russia  iron  box  for  holding  plates,  etc.,  during  sterilization  in  dry  heat.    (Abbott.) 

7.  Glass  benches  for  supporting  plates  (Fig.  17). 


64 


THE  CULTIVATION  OF  BACTERIA. 
FIG.  17. 


Glass  benches  for  supporting  plates. 


8.  Graduated   measuring    cylinders    of  100  and    1000  c,c. 
pacity  (Fig.  18). 

9.  Graduated  pipettes  of  1  c.c.  capac 
and  10  c.c.  divided  into  c.c.  (Fig.  19). 


capacity  (Fig.  18). 

9.  Graduated  pipettes  of  1  c.c.  capacity  divided  into  tenths, 


FIG.  18. 


FIG.  19. 


FIG.  20. 


FIG.  21. 


Measuring  cylinder. 


Graduated  pipette. 


Sternberg  bulb. 


Bulb-pipette, 


10.  Sternberg  bulbs  (Fig.  20). 

11.  Bulb-pipettes  (Fig.  21). 

12.  Petri's  double  dishes  (Fig.  22). 

FIG.  22. 


Petri's  double  dish,  now  generally  used  instead  of  plates.    (Abbott.) 


THE   UTENSILS  OF  THE  CULTIVATION  OF  BACTERIA.  65 
FIG.  23.  FIG.  24.  FIG.  25. 


Moist  chamber  with  a  knob  on  the  Wooden  filter-stand.       Iron  stand  with 

upper  dish.  rings. 

FIG.  26. 


Iron  tripod  with  water-bath. 
FIG.  27. 


Wire  baskets. 
5— M   B. 


66  THE  CULTIVATION  OF  BACTERIA. 

FIG.  28. 


Pinchcock. 


13.  Moist  chambers  for  potato  and  plate  cultures  (Fig.  23). 

14.  Glass  funnels  of  different  sizes,  1,  4,  and  8  ounces. 

15.  Wooden  filter-stands  (Fig.  24). 

FIG.  30. 


FIG.  29. 


Fermentation-tube  on  left  side;  ordin- 
ary tube  on  right  side, 


Anatomical  jar  for  collecting 
blood. 


16.  Iron  stands  with  rings  and  clamps  (Fig.  25). 

17.  Iron  tripods  with  water-baths  (Fig.  26). 

18.  Test-tube  racks  of  any  standard  design. 


QUESTIONS.  67 

19.  Square  and  round  iron  wire  baskets  for  sterilizing  test- 
tubes  (Fig.  27). 

20.  Perforated  tin  buckets  for  sterilizing  potatoes. 

21.  Pinchcocks  (Fig.  28)  for  holding  test-tubes. 

22.  Fermentation-tubes  (Fig.  29). 

FIG.  31. 


Wolf  huegel's  ruled  plate  for  counting  colonies. 

23.  A  2-  and  a  4-liter  anatomical  jar,  with  tightly  fitting 
cover,  for  the  collection  of  blood-serum  (Fig.  30). 

24.  Wolf  huegel's  ruled  plates  for  counting  colonies  (Fig.  31). 

25.  Bunsen  burners  of  different  sizes. 

QUESTIONS. 

What  is  meant  by  a  culture-medium  ? 

What  is  meant  by  the  sterilization  of  culture-media  ? 

How  is  milk  prepared  as  a  culture-medium  ? 

How  is  milk  sterilized  when  used  for  a  medium  ? 

Why  is  tincture  of  litmus  added  to  milk  medium  ? 

How  does  milk  help  in  differentiating  between  different  species  of  bacteria  ? 

What  is  the  process  of  making  blood-serum  ? 

.How  is  blood-serum  sterilized? 

What  is  Loeffler's  modification  of  the  blood-serum  method  ? 

What  other  animal  fluids  are  occasionally  used  as  culture-media? 

What  does  Pasteur's  solution  consist  of? 

How  would  you  prepare  bouillon  for  cultures  ? 

What  are  the  principal  solid  cultures? 

How  would  you  prepare  a  nutrient  gelatin? 

How  is  nutrient  gelatin  sterilized  ? 

What  are  the  advantages  of  using  gelatin  as  a  culture  ?  What  are  its  dis- 
advantages? 

How  would  you  prepare  nutrient  agar?  What  is  difficult  in  the  prepara- 
tion of  it?  How  would  you  sterilize  it?  What  are  its  advantages  and  dis- 
advantages when  compared  to  gelatin  ? 

How  is  agar-glycerin  made  ? 

How  is  agar-gelatin  made  ? 

What  is  the  best  way  to  filtrate  agar  ? 


68     INOCULATION  OF  CULTURE-MEDIA    WITH  BACTERIA. 

Give  Koch's  method  of  preparing  potatoes  for  a  culture?    What  are  the 
precautions  necessary  for  protecting  the  potato  from  external  germs  ? 
How  would  you  prepare  a  potato  test-tube  culture  ? 
How  would  you  prepare  Dunham's  solution? 
How  are  glucose-,  lactose-,  and  saccharose-bouillon  prepared  ? 


CHAPTEK  IV. 

THE   INOCULATION    OF    CULTURE-MEDIA    WITH 
BACTERIA. 

THE  METHOD  OF  INOCULATING  FLUID  MEDIA. 

FOE  the  purpose  of  cultivating  bacteria  in  liquid  media  it 
is  only  necessary  to  introduce  the  smallest  possible  particle 
containing  the  bacteria,  into  the  media,  by  means  of  a  steril- 
ized platinum  needle  (Fig.  15).  For  this  procedure  the 
culture-tube  is  held  slightly  inclined  between  the  thumb  and 
fingers  of  the  left  hand,  its  cotton  plug  removed  by  a  twist- 
ing motion  and  placed  between  the  backs  of  the  third  and 
fourth  fingers  of  this  hand,  being  careful  not  to  touch  that  por- 
tion of  the  cotton  which  fits  inside  of  the  tube.  The  inoculating 
needle  is  rapidly  introduced  into  the  liquid  and  gently  stirred 
around,  after  which  the  cotton  plug  is  replaced,  and  the  plat- 
inum needle  sterilized  by  heating  to  redness  in  the  flame. 

It  is  good  practice  to  accustom  one's  self  never  to  take  up 
a  platinum  needle,  whether  it  is  known  to  be  inoculated  or  unin- 
oculated,  without  first  sterilizing  it  by  heating  it  to  redness  in  the 
flame,  and  allowing  it  to  cool  for  a  few  seconds  before  taking 
up  with  it  the  inoculating  material.  The  bacteria  might  other- 
wise be  destroyed  by  the  heat.  Again,  after  use  the  needle  should 
always  be  sterilized  in  the  same  manner  before  putting  it  down. 

THE   METHODS  OF  INOCULATING  SOLID    MEDIA. 

1.  For  the  inoculation  of  potatoes  and  other  solid  media 
not  hereafter  mentioned,  it  is  only  necessary  to  streak  the 
surface  of  the  medium  with  a  platinum  needle  or  other  instru- 
ment which  has  been  dipped  into  or  which  contains  a  small 


THE  METHODS  OF  INOCULATING  SOLID  MEDIA.     69 

fragment  of  the  contaminating  material,  care  having  been 
taken  beforehand  to  sterilize  thoroughly  the  needle  or 
instrument. 

2.  Gelatin  culture-tubes  are  inoculated  in  one  of  three  ways  : 

a.  Stab   culture,   made    by   puncturing   the   centre  of  the 
solidified   gelatin   mass  with   a   platinum  needle  previously 
charged  with  the  bacteria. 

b.  Slant  cultures,  made  by  gently  passing  over  the  surface  of 
the  medium   the   inoculating  needle ;    for  this   purpose  the 
gelatin  is  made  to  solidify  with  the  tube  in  an  inclined  posi- 
tion, so  as  to  give  a  larger  surface  for  inoculation. 

c.  Plate   cultures,  made   by  inoculating  the  gelatin  mass, 
which  has  been  previously  liquefied  by  submitting  it  to  a 
temperature  of  30°  C.,  with  a  platinum  needle  or  loop  as 
for  liquid  cultures,  and  pouring  the  liquid  mass  rapidly  and 
evenly  on  a  sterilized  glass  plate,  allowing  it  to  solidify  well 
protected  from  dust. 

The  plate  method,  introduced  by  Koch,  is  of  great  value 
for  the  separation  and  isolation  of  bacteria.  In  this  manner 
each  bacterium  introduced  into  the  liquefied  gelatin  is  fixed  by 
the  hardening  of  the  gelatin  and  develops  as  a  separate  colony, 
the  number  of  colonies  being,  as  a  rule,  equal  to  the  number 
of  bacteria  originally  introduced. 

Each  colony  grows  in  its  own  peculiar  way,  because  each 
species  has  a  definite  way  of  growing  in  gelatin.  This 
method  therefore  not  only  serves  for  the  separation  of  the 
bacteria  themselves,  but  also  enables  us  to  recognize  one 
species  from  another.  Indeed,  the  classical  method  of  Koch 
for  the  separation  and  isolation  of  bacteria,  though  modified 
in  some  particulars,  has  not  been  essentially  changed  since  its 
introduction.  It  is  as  follows  : 

Three  sterilized  gelatin  culture-tubes  about  a  third  full  and 
containing  about  10  c.c.  of  the  medium  are  liquefied  by  being 
submitted  to  a  temperature  of  30°  C.  Tube  I.  is  inoculated 
with  one  or  two  loopfuls  of  the  platinum  needle  from  the 
contaminated  substance,  its  cotton  plug  replaced,  and  the 
contents  well  shaken.  After  sterilizing  the  platinum  needle 
one  or  two  loopfuls  from  tube  No,  I,  are  introduced  into  tube 


70     INOCULATION  OF  CULTURE-MEDIA    WITH  BACTERIA 

No.  II. ;  both  tubes  are  again  plugged,  and  tube  No.  II.  is 
in  turn  well  shaken.  The  platinum  needle  is  again  sterilized, 
and  finally  one  or  two  loopfuls  from  tube  No.  II.  are  intro- 
duced into  Tube  No.  III.,  and  the  contents  of  the  latter  well 
stirred. 

The  three  tubes  are  kept  on  a  water-bath  at  a  temperature 
of  between  25°  and  30°  C.,  so  as  to  keep  the  mass  liquid. 
Meanwhile  three  sterilized  glass  plates  are  arranged  on  three 
cooling-stages,  as  depicted  in  Fig.  32.  The  gelatin  from  each 

FIG.  32. 


Levelling-tripod  with  glass  cooling-chamber  for  plates. 

of  the  tubes  I.,  II.,  III.  is  slowly  and  evenly  poured  over 
the  surface  of  the  plates,  correspondingly  designated  as  No. 
I.,  II.,  III.,  and  allowed  to  solidify.  It  is  necessary  during 
the  pouring  and  solidification  of  the  gelatin  on  the  plates  that 
they  be  carefully  protected  by  a  cover  against  the  dust  and 
bacteria  from  without. 

After  solidification  is  perfect  the  plates  are  transferred  into 
culture-dishes  placed  on  glass  benches  (Fig.  17),  and  properly 
labelled. 

To  harden  the  gelatin  more  rapidly,  ice  or  iced  water  is 
generally  kept  in  the  lower  dish  of  the  cooling-stage.  To  insure 


THE  METHODS  OF  INOCULATING  SOLID  MEDIA.      71 

evenness  of  surface  in  the  gelatin  on  the  glass  plates  a  levelling- 
tripod  is  used,  as  seen  in  Fig.  32.  This  tripod  is  easily  set 
by  means  of  a  levelling  glass. 

Results. — In  this  manner  each  of  the  separate  bacteria  con- 
tained in  the  media  will  develop  as  separate  colonies  on  the 
gelatin  plates.  Plate  I.,  made  from  tube  I.,  will  contain  a 
large  number  of  bacteria ;  plate  II.  will  contain  the  bacteria 
in  much  smaller  number ;  and  plate  III.  will  contain  only  a 
few  colonies,  well  separated  ;  and  in  this  way  the  characteristic 
growth  of  the  separate  colonies  and  their  action  on  gelatin 
may  be  carefully  followed  out  and  studied.  By  this  means 
also  the  observer  may  under  a  magnify  ing-glass  with  a  fine 
sterile  platinum  needle  pick  out  the  individual  colonies  and 
inoculate  fresh  tubes,  and  so  obtain  pure  cultures  of  any  liv- 
ing organism. 

3.  Plate  cultures  of  agar  are  made   in  the  same  way,  but 
require  more  care  in  their  preparation,  as  agar  does  not  melt 
at  a  temperature  below  42°  C.,  and  solidifies  again  at  a  tem- 
perature of  39°  or  40°  C.,  so  that  after   inoculation   in   the 
liquid  state  the  tubes  must  be  kept  in  a  water-bath  between 
40°  and  42°  C.  until  they  are  poured  into  the  plates.     The 
agar  plates,   however,   may  be  incubated  at  blood  tempera- 
ture (37°  C.),  and  the  growth  of  bacteria  noted. 

In  recent  years  small  double  dishes,  known  as  Petri's  culture- 
dishes  (Fig.  22),  have  been  introduced  to  take  the  place  of 
the  plates.  For  the  inoculation,  liquefied  gelatin  or  agar  is 
poured  into  the  lower  dish,  and  this  is  quickly  covered  by 
the  upper  dish.  For  this  method  no  levelling  apparatus  and 
cooling-stage  are  required. 

For  counting  the  colonies  in  the  plates  the  apparatus  of 
Wolfhuegel  (Fig.  31)  has  been  adopted. 

This  consists  of  regularly  lined  glass  plates,  divided  into 
squares  and  arranged  as  seen  in  the  figure.  By  placing  the 
culture-plate  under  the  lined  plate  it  is  easy  to  ascertain  the 
number  of  colonies  contained  in  each  of,  for  example,  ten 
squares,  and  by  a  simple  process  of  multiplication  the  number 
of  colonies  in  the  whole  plate. 

4.  Instead  of  pouring  out  the  liquid  gelatin  from  the  tubes 


72     INOCULATION  OF  CULTURE-MEDIA    WITH  BACTERIA. 

into  plates  after  inoculation,  Esmarch  rolls  these  tubes  in  a 
vertical  position  until  the  gelatin  is  completely  solid.  This  is 
hastened  by  rolling  the  tubes  as  shown  in  Fig.  33.  By  this 
excellent  method  there  is  less  likelihood  of  contamination 
than  by  the  plate  method. 

5.  Gelatin  and  especially  agar  plates  are  occasionally  made 
in  a  different  way.  The  liquid  medium  is  poured  on  glass 
plates  and  allowed  to  solidify  before  inoculation.  When  well 
hardened  the  surface  is  streaked  with  the  inoculating  material 

FIG.  33. 


Demonstrating  Booker's  method  of  rolling  Esmarch  tubes  on  a  block  of  ice. 

on  a  platinum  needle.  In  this  way  the  colonies  grow  along 
the  streak  much  more  superficially  than  they  do  in  the  ordi- 
nary plate  method. 

6.  Agar  and  blood-serum  slant  cultures  are  made  in  the  same 
manner  as  similar  cultures  on  gelatin.  They  have  one 
advantage  over  the  gelatin  culture  by  reason  of  being  able  to 
withstand  the  temperature  of  the  incubator,  37°  C. 

THE  CULTIVATION  OF  ANAEROBIC  BACTERIA. 

Exclusion  of  oxygen  is  absolutely  necessary.  For  this  pur- 
pose a  number  of  methods  have  been  suggested  and  used, 
§ome  of  which  require  special  and  elaborate  apparatus.  All 


THE  CULTIVATION  OF  ANAEROBIC  BACTERIA.       73 


the  foregoing  methods  of  inoculation  and  cultivation  are  avail- 
able only  for  the  aerobic  bacteria. 

1.  Cultivation  of  Tetanus  Bacillus. — The  following  method 

FIG.  34. 


FIG.  35. 


Jar  for  anaerobic  cultures. 
FIG.  36. 


Small  incubator. 


Mercurial  thermo-regulator. 


has  been  found  very  useful  for  the  cultivation  of  this  strictly 
anaerobic  bacillus,  aud  answers  all  purposes  in  the  author's 
opinion, 


74    INOCULATION  OF  CULTURE-MEDIA    WITH  BACTERIA. 

A  culture-tube  three-fourths  full  of  the  medium  is  heated 
to  the  boiling-point  and  allowed  to  cool  to  a  temperature  in 
the  neighborhood  of  40°  C.;  then  a  platinum  needle  charged 

FIG.  37. 


Incubator  used  in  bacteriological  W9rk :  a  represents  the  incubator  set  up  and 
containing  a  cage  of  tubes  and  a  Petri  dish ;  6,  represents  a  vertical  section  of 
the  incubator  and  displays  the  water-chamber,  inner  chamber,  walls,  vents,  ther- 
mometer, valve,  etc. 

with  the  material  for  inoculation  is  dipped  down  to  the  bot- 
tom of  the  tube.  With  fluid  media,  immediately  thereafter 
a  layer  of  paraffin  oil  is  poured  on  the  surface  of  the  liquid 


QUESTIONS.  75 

before  introduction  of  the  cotton  plug.  With  solid  media, 
the  fluid  is  allowed  to  solidify  after  inoculation,  and  after 
solidification  the  paraffin  oil  is  poured  on  the  surface  of  the 
medium.  This  effectually  shuts  out  the  oxygen,  and  as  a  rule 
allows  a  luxuriant  growth  of  anaerobic  bacteria. 

2.  Special  apparatus,  with  oxygen  replaced  by  hydrogen,  is 
also  used  for  the  cultivation  of  anaerobic  bacteria. 

The  Incubator  and  The  Thermostat. 

For  the  purpose  of  growing  bacteria  it  is  often  necessary 
or  desirable  to  obtain  a  constant  temperature.  The  ordinary 
body-temperature,  37°  C.,  is  the  most  favorable  for  the  growth 
of  the  pathogenic  bacteria.  Apparatus  especially  constructed 
for  maintaining  a  constant  temperature  are  known  as  incuba- 
tors or  thermostats.  They  are  generally  made  of  double- 
walled  metal,  and  contain  water,  which  by  means  of  a  gas-jet 
at  the  bottom  of  the  apparatus  may  be  kept  at  a  constant 
temperature  (Fig.  37). 

For  regulating  the  gas-supply  and  to  maintain  the  constant 
temperature,  instruments  known  as  thermo-regulators  are 
used.  A  number  of  these  are  highly  complicated,  but  for 
ordinary  work  is  recommended  Reichert's  or  Dunham's  mer- 
curial thermo-regulator  (Fig.  35). 

QUESTIONS. 

How  do  you  inoculate  a  fluid  culture-medium  ? 

How  do  you  inoculate  potato  cultures? 

In  how  many  ways  may  gelatin  tubes  be  inoculated? 

What  is  meant  by  a  stab-culture  ?    By  a  slant  culture? 

How  are  plate  cultures  made? 

Describe  the  method  of  making  plate  cultures  according  to  Koch.  How 
sire  agar  plate  cultures  made? 

How  are  colonies  on  plates  counted? 

Describe  the  method  of  making  cultures  in  Petri  dishes? 

How  are  Esmarch's  culture-tubes  made? 

Give  a  good  method  for  the  cultivation  of  anaerobic  bacteria.  What  is  an 
incubator? 

What  is  a  thermo-regulator? 


76     STERILIZATION,    DISINFECTION,  AND  ANTISEPSIS. 

CHAPTER  V. 

STERILIZATION,   DISINFECTION,   AND  ANTISEPSIS 

Definitions. — The  freeing  of  substances  from  the  live  bacteria 
they  may  contain  or  that  may  have  collected  on  their  surfaces 
is  called  sterilization.  This  is  accomplished  either  by  means 
of  heat  or  the  use  of  chemicals. 

Erroneously  sometimes  the  term  sterilization  is  used  to 
indicate  the  destruction  of  bacteria  by  the  application  of  heat, 
the  term  disinfection  being  used  then  for  their  destruction  by 
chemical  agents. 

To  disinfect  a  substance  is  to  destroy  in  it  or  on  it  all  the 
harmful  or  infectious  bacteria,  without  necessarily  killing  all 
the  living  bacteria. 

THE  METHODS  OF  STERILIZATION. 

I.  Substances  chemically  sterilized  are   unfit  for  bacterial 
culture,  except   in  very  rare   instances   when   the   chemical 
agents  are  very  volatile. 

II.  In  laboratory  work,  therefore,  where  the    aim  is  the 
cultivation  of  bacteria,  heat  is  the  only  method  of  sterilization 
used.     This  is  applied  either  in  the  form  of  dry  heat  or  moist 
heat  (steam). 

1 .  All  substances  which  may  be  passed  through  the  Bunsen 
flame  and  heated  red-hot  are  usually  sterilized  in  this  manner. 

2.  Other  implements,  such  as  instruments  and  glassware, 
which  would  be  injured  by  the  direct  flame,  but  withstand  con- 
siderable heat,  are  sterilized  by  dry  heat  in  an  oven  at  a 
temperature  of  160°  to  180°  C.  for  an  hour. 

3.  For  culture-media,  one  resorts  to  sterilization  by  steam, 
except  in  rare  instances,  when  filtration  under  pressure  through 
unglazed  porcelain  is  considered  sufficient. 

Experience  has  taught  that  steam  at  a  temperature  of 
100°  C.  will  kill  all  known  bacteria  and  their  spores  within 
an  hour,  and  Pasteur  has  demonstrated  that  steam  under  a 
pressure  of  two  or  three  atmospheres,  at  about  130°  C.,  will 


THE  METHODS  OF  STERILIZATION. 


77 


destroy  all  known  bacteria  and  their  spores  within  fifteen  or 
twenty  minutes.  The  sterilization  of  culture-media,,  then,  is 
usually  done  by  the  means  of  steam,  with  or  without  pressure. 

4.  In  some  instances,  when  exposure  to  steam  for  one  hour 
would  be  prejudicial  to  the  medium,  what  is  known  as  dis- 
continuous or  fractional  sterilization  is  resorted  to.  The 
medium  is  steamed  during  three  consecutive  days  for  twenty 
minutes  each  time  ;  during  the  interval  it  is  kept  in  favorable 

FIG.  38. 


FIG.  39. 


Laboratory  hot-air  sterilizer. 


Rose-burner. 


conditions  for  the  development  of  bacteria.  In  this  manner 
the  first  heat  destroys  all  the  fully  formed  bacteria  that  may 
exist.  The  favorable  temperature  in  the  interval  between 
the  first  and  second  heatings  allows  all  the  spores  contained 
in  the  medium  which  may  not  have  been  destroyed  in  the 
first  heating  to  develop  into  fully  formed  germs,  which  are 
destroyed  by  the  second  application  of  steam  on  the  ensuing 
day.  The  application  of  heat  on  the  third  day  is  to  make 


78     STERILIZATION,  DISINFECTION,  AND  ANTISEPSIS. 


sure  of  the  destruction  of  all  spores  which  may  have  resisted  the 
two  previous  applications.  It  has  been  demonstrated  that 
this  sterilization  is  very  effective  and  complete,  and  substances 
which  have  undergone  it  may  be  kept  indefinitely  bacteria- 
free  thereafter. 

5.  Another  method  of  fractional  sterilization  is  occasionally 
used  for  certain  media  which  cannot  withstand  the  tempera- 
ture of  boiling  water  without  deteriorating.  It  consists  in 

FIG.  40. 


Steam  sterilizer,  pattern  of  Koch.    (Abbott.) 

submitting  the  medium  to  a  temperature  of  68°  to  70°  C.  for 
from  two  to  three  hours  during  seven  consecutive  days.  This 
method  is  necessarily  very  imperfect,  and  though  successful 
in  those  cases  in  which  no  spores  have  to  be  destroyed  and 
where  none  of  the  bacteria  contained  in  the  medium  are  of 
the  pyrogenic  variety,  it  cannot  but  have  a  very  limited 
application,  and  media  so  sterilized  should  not  be  used  for 


THE  METHODS  OF  STERILIZATION. 


79 


culture  purposes,  except  after  they  have  been  submitted  for 
several  days  to  the  temperature  of  the  human  body  in  the 
thermostat  and  remained  sterile  during  this  control-test. 

6.  For  generating  dry  heat  for  the  sterilization  of  glass- 
ware, implements,  and  instruments  used  in  making  cultures, 
the  hot-air  oven  and  rose-burner  are  most  usually  employed, 
and  a  temperature  usually  of  180°  C,  maintained  in  the  oven 
for  one  and  one-half  hours  (Figs.  38  and  39). 

FIG.  41. 


Arnold  steam  sterilizer.    (Abbott.) 

The  oven  consists  of  a  double-walled  metallic  box,  with  a 
double-walled  front  door,  with  a  copper  bottom,  the  sides  and 
door  encased  in  asbestos  boards.  The  heat  is  applied  by 
means  of  a  rose  gas-burner  (Fig.  39)  to  the  bottom  of  the  box. 
The  objects  to  be  sterilized  are  put  upon  perforated  metallic 
shelves  in  the  box.  In  the  top  of  the  apparatus  are  two  per- 
forated openings  for  the  insertion  of  the  thermometers.  In 
this  portion  the  oven  is  also  provided  with  a  perforated  slid- 
ing window  to  allow  escape  of  the  overheated  air. 


80     STERILIZATION,  DISINFECTION,  AND  ANTISEPSIS. 


7.  For  applying  moist  heat,  bacteriologists  commonly  use 
a.  Koch's  apparatus  (Fig.  40).  b.  Arnold's  apparatus  (Fig.  41). 

8.  For  the   application  of  steam  under  pressure  preference 
is   felt   for   the    autoclave    of   Chamberlain   or   of    Wiesnegg 
(Fig.  42). 

9.  For  discontinuous  fractional  sterilization  at  a  low  tem- 

FIG.  42. 


JL 


A  B 

Autoclave,  pattern  of  Wiesnegg:  A,  external  appearance;  B,  section.    (Abbott.) 

perature  the  apparatus  in  more  general  use  is  the  blood-serum 
sterilizer  of  Koch  (Fig.  43). 

10.  Filtration  under  pressure  through  unglazed  porcelain,  the 
pores  of  which  are  too  small  to  allow  bacteria  to  go  through, 
will  render  certain  liquid  substances  sterile  or  bacteria-free. 
This  method  is  made  use  of  in  the  case  of  certain  pathogenic 
bacteria  which  secrete  soluble  poisons  which  we  desire  to 
separate  from  the  bacteria  themselves.  The  Chamberlain 


THE  METHODS  OF  DISINFECTION. 


81 


filter  of  unglazed  Sevre  porcelain  is  the  only  one  to  be  recom- 
mended for  this  purpose. 


FIG.  43. 


Chamber  for  sterilizing  and  solidifying  blood-serum.    (Koch.) 

THE  METHODS  OF  DISINFECTION. 

I.  Disinfection  or  destruction  of  infectious  bacteria  may  with 
certainty  be  accomplished  by  heat,  and  indeed  in  the  labora- 
tory when   the    total   destruction  of  the   bacteria  is  desired 
with  the  material  containing  them  heat  is  the  most  effective 
measure. 

In  other  cases,  however,  when  the  destruction  of  the  bac- 
teria themselves  and  the  preservation  of  the  contaminated  sub- 
stance are  desired,  recourse  must  be  had  to  such  measures  as 
will  destroy  the  bacteria  alone. 

Substances  which  are  able  to  destroy  bacteria  or  their  \ 
spores  are  known  as  germicides  or  disinfectants,  and  those  J 
substances  which  retard  bacterial  growth  are  called  antiseptics.  / 

II.  In   the   use  of  chemicals  for   disinfecting,   one   should 
always  bear  in  mind  that  their  mode  of  action  is  not  a  cata- 
lytic one,  but  that  they  owe  their  virtue  to  the  power  of 

6— M.  B. 


82     STERILIZATION,  DISINFECTION,  AND  ANTISEPSIS. 

forming  definite  chemical  compounds  with  the  bacteria  cells, 
which  are  thus  rendered  innocuous.  They  must,  therefore, 
come  into  direct  contact  with  the  bacteria  themselves,  and  in 
the  combination  so  formed  they  and  the  bacteria  change  their 
chemical  properties. 

In  the  choice  of  chemical  disinfectants,  one  must  be  guided 
by  the  species  of  bacteria  to  be  destroyed,  by  the  number  of 
these  bacteria,  by  the  nature  of  the  media  containing  the 
germs,  by  the  substances  to  be  disinfected,  and  by  the  quan- 
tity of  material  to  be  disinfected.  All  chemicals  so  used 
must  be  of  a  definite  strength,  and  must  be  made  to  act  for 
a  specified  time,  the  quantity  and  strength  of  the  disinfec- 
tant and  the  time  varying  with  the  nature  of  the  chemical 
and  the  species  of  bacteria  to  be  destroyed. 

To  test  the  germicidal  power  of  substances,  the  following  is 
a  convenient  method :  To  young  bouillon  cultures  of  the 
bacteria  to  be  acted  upon  sufficient  of  the  chemical  to  be 
tested  is  added  to  make  the  proper  dilution,  and  at  given 
intervals  of  time  a  few  droplets  of  this  mixture  are  inocu- 
lated on  sterile  agar  and  gelatin  tubes  and  the  result  carefully 
noted — for  instance,  supposing  it  is  intended  to  test  the  germi- 
cidal power  of  carbolic  acid  toward  some  pus  organism.  To 
9.90  c.c.  of  a  bouillon  culture  of  a  pus  germ  in  a  test-tube 
0.10  c.c.  of  carbolic  acid  is  added,  making  the  mixture  a 
1  per  cent,  carbolic  acid  dilution.  The  tube  is  well  shaken, 
and  at  the  end  of  one  minute  a  few  droplets  of  the  mixture 
are  taken  on  a  sterile  platinum  needle  and  inoculated  on  a 
fresh  tube  of  gelatin  or  agar,  another  fresh  agar  tube  is 
inoculated  with  the  mixture  in  the  same  manner  after  two 
minutes,  another  again  after  five  minutes,  and  a  fourth  in 
fifteen  minutes,  a  fifth  in  a  half  hour,  a  sixth  in  an  hour, 
and  a  seventh  in  two  hours.  The  growth  in  the  inoculated 
tubes  would  indicate  the  action  of  1  per  cent,  carbolic  acid 
upon  the  given  pus  germ  in  the  specified  time.  The  same 
process  is  repeated,  using  a  2  per  cent,  dilution  of  the  car- 
bolic acid,  and  next  a  3  per  cent.,  a  4  per  cent.,  and  a  5  per 
cent,  dilution.  Higher  than  5  per  cent,  solutions  of  carbolic 
acid  are  not  possible  with  any  but  water  too  hot  for  this  work. 


THE  METHODS  OF  ANTISEPSIS.  83 

In  other  substances  a  10  percent,  dilution  would  be  the  last 
step  of  the  test.  Thus  are  obtained  results  which  are  fixed 
and  definite,  stating  positively  the  strength  of  disinfection 
used  and  the  time  of  its  application. 

Cautions. — In  conducting  these  experiments  one  must 
remember  always  that  the  growth  of  bacteria  on  the  solid 
tubes  may  be  considerably  retarded  by  the  action  of  the  dis- 
infectants, and  one  must  not  accept  the  result  as  conclusive 
unless  those  cultures  have  been  kept  under  observation  for  a 
considerable  length  of  time.  Again  it  is  proper,  whenever 
practicable,  to  conduct  the  experiments  at  the  temperature  of  the 
human  body,  37°  C.,  as  experience  has  demonstrated  that  at 
this  and  higher  temperature  the  disinfectant  power  of  chemicals 
is  increased. 


THE  METHODS  OF  ANTISEPSIS. 

Substances  that  retard  the  growth  of  bacteria  without, 
however,  destroying  them  are  called  antiseptics.  It  is  clear 
that  all  disinfectants  when  used  in  a  more  diluted  form,  or 
when  allowed  to  act  for  a  shorter  space  of  time  than  is  re- 
quired for  them  to  show  their  germicidal  power,  act  as  anti- 
septics. 

I.  The  Common  Disinfectants. 

Carbolic  acid,  strength  3  to  5  per  cent.,  efficient  in  one  hour. 

Bichloride  of  mercury,  solution  1  :  1000  or  1  :  500,  acts  from 
within  a  few  minutes  to  a  half-hour. 

Chlorinated  lime,  containing  free  chlorine,  is  an  efficient 
gormicide  in  an  hour's  time  in  the  strength  of  from  5  to 
1 0  per  cent. 

Boiling  water,  to  which  2  or  3  per  cent,  sodium  carbonate 
is  added,  is  an  efficient  germicide  in  an  hour. 

Sulphur  dioxide  gas,  when  used  dry,  has  little  or  no  disin- 
fectant power,  and  bacteria  have  been  able  to  withstand  an 
atmosphere  containing  from  10  to  12  per  cent,  of  this  gas  for 
several  hours.  In  the  presence  of  moisture,  however,  it 


84     STERILIZATION,  DISINFECTION,  AND  ANTISEPSIS. 

forms  sulphurous  acid,  and  is  then  an  efficient  germicide  in  as 
low  a  percentage  as  4  or  5  per  cent. 

Formalin,  in  2  to  5  per  cent,  strength,  or  as  formaldehyde 
gas,  is  a  powerful  germicide. 

A  number  of  other  substances  have  been  recommended, 
and  have  been  used  as  germicides,  but  the  above  are  of  more 
general  and  practical  value. 

II.  Enumeration  of  the  common  antiseptics  would  be  too 
lengthy  to  be  stated  in  a  book  of  this  kind. 

In  conclusion,  one  should  bear  in  mind  that  chemical  agents 
must  act  directly  on  the  bacteria  cells  themselves — that  is, 
they  must  penetrate ;  and  that  they  are  the  more  efficient  the 
more  quickly  they  form  chemical  compounds  with  those  cells 
— or,  as  usually  expressed,  the  more  penetrating  power  they 
possess — and  anything  that  interferes  with  this  chemical  com- 
bination interferes  with  the  action  of  the  disinfectant. 

QUESTIONS. 

What  is  meant  by  sterilization  ?    By  disinfection  ? 

At  what  temperature  and  how  long  does  it  take  dry  heat  to  sterilize? 

How  long  does  it  take  live  steam  to  sterilize  ? 

How  long  does  it  take  steam  under  pressure  to  sterilize? 

How  are  implements  in  the  bacteriological  laboratory  sterilized? 

How  are  culture-media  sterilized  ? 

What  is  discontinuous  or  fractional  sterilization  by  steam,  and  how  is  it 
accomplished  ? 

How  is  sterilization  at  a  low  temperature  effected?  When  is  it  used? 
What  are  its  disadvantages  ? 

What  forms  of  apparatus  are  generally  used  to  generate  steam  for  sterili- 
zation ? 

What  are  the  only  filters  which  can  remove  bacteria  from  liquid  media? 

What  are  germicides  or  disinfectants? 

What  are  antiseptics? 

How  would  you  test  the  value  of  a  germicide  ? 

What  chemicals  are  generally  used  as  disinfectants? 

State  the  value  of  carbolic  acid  as  a  disinfectant ;  of  bichloride  of  mercury  ; 
of  the  chlorinated  lime ;  of  sodium  carbonate ;  of  sulphur  dioxide,  and  of 
formaldehyde. 


THE  INOCULATION  OF  ANIMALS  AND  THEIR  STUDY.    85 

CHAPTEE  VI. 

THE   INOCULATION   OF  ANIMALS  AND    THEIR    STUDY. 

THE  INOCULATION  OF  ANIMALS. 

ITS  purposes  are  to  differentiate  between  bacteria.  In  order 
to  study  their  virulence  it  is  often  necessary  to  test  their  action 
on  animals. 

In  the  laboratory  the  smaller  animals,  such  as  mice,  rats, 
guinea-pigs,  and  rabbits,  are  chiefly  used. 

Technic. — The  inoculation  is  made  in  a  number  of  ways, 
depending  on  the  species  of  the  bacteria,  the  nature  of  its 
toxin,  and  the  rapidity  of  action  desired. 

The  Various  Methods  of  Inoculation  of  Animals. 

1.  Sometimes,  though   rarely,  the  inoculation  is  made  by 
rubbing  a  solid  or  liquid  culture  over  the  abraded  epidermis, 
very  much  in  the  same  manner  as  vaccine  is  introduced  into 
the  human  subject. 

2.  Subcutaneous    inoculation — that  is,  into  the   connective 
tissue   under   the  skin — is  an  important  method.     For  this 
purpose  the  hair  is  shaved  from  part  of  the  back  or  abdomen,- 
the  skin  well  washed  and  disinfected  as  well  as  it  may  be, 
with  a  5  per  cent,  carbolic  acid  solution.     Then  the  skin  is 
seized  with  a  pair  of  sterilized  forceps,  and  with  a  sterilized 
scalpel  a  small  nick    is  .made  into  it,   after  which   a  small 
sterilized  pair  of  scissors   is  introduced  in  the  areolar  tissue 
and  a  pocket  made  for  some  little  distance  into  this  tissue. 
Into  this  pocket  the  inoculating  material  or  bacterial  culture 
(especially  when  a  solid  culture  has  been  employed)  is  intro- 
duced, by  means  of  a  sterilized  forceps  or  a  platinum  loop, 
care  being  taken  to  avoid  touching  the  edges  of  the  wound  with 
the  instrument. 

If  a  liquid  culture  is  being  used,  particularly  in  appreciable 
quantity,  it  may  be  introduced  subcutaneously  by  means  of  a 
hypodermatic  syringe  and  needle  well  sterilized  beforehand. 


86    THE  INOCULATION  OF  ANIMALS  AND  THEIR  STUDY. 

Guinea-pigs  and  rabbits  are  usually  inoculated  in  the  abdom- 
inal wall ;  rats  or  mice  in  the  loose  tissue  at  the  root  of  the 
tail. 

Animal  Holders. — Various  instruments  have  been  devised 

FIG.  44. 


The  Voges  holder  for  guinea-pigs.    (Abbott.) 

for  keeping  animals  quiet  during  this  operation,  the  most  use- 
ful of  which  are  :  Voges'  guinea-pig-holder  (Fig.  44) ;  Kitasato's 
mouse-holders  (Fig.  45)  ;  and  the  basket  mouse-holder  (Fig.  46). 
3.  Intravenous  injection  or  inoculation  into  the  circulation 


THE  INOCULATION  OF  ANIMALS. 


87 


consists  in  injecting  directly  into  the  veins  of  the  animal  in 
the  direction  of  the  circulation,  the  material  to  be  inoculated. 
Necessarily  the  material  used  must  be  a  liquid,  and  the  injec- 
tion must  be  done  slowly  and  with  precaution.  Intravenous 
injection  is  used  especially  in  rabbits.  The  most  convenient 
point  of  injection  is  into  the  vein  of  the  ear  known  as  the 
posterior  auricular  vein,  which  is  easily  penetrated  from  the 
dorsal  surface  of  the  ear,  where  it  lies  superficial  and 


FIG.  45. 


Kitasato's  mouse-holder. 
(Abbott.) 


Mouse-holder,  with  mouse  in  proper  position. 
(Abbott.) 


imbedded  firmly  in  the  areolar  tissue.  For  the  pur- 
pose of  making  these  injections  an  ordinary  hypodermatic 
syringe  with  the  needle  used  for  morphine  injections  in  man 
is  employed.  All  that  is  required  is  that  both  syringe  and 
needle  be  sterilized.  The  mode  of  procedure  is  as  follows : 
The  rabbit  is  firmly  held  by  an  assistant.  The  ear  chosen 
is  taken  between  the  thumb  and  forefinger  of  the  left  hand 
and  after  washing  and  sterilizing  the  skin  as  thoroughly  as 
possible,  the  vein  on  the  posterior  edge  of  the  ear  is  sought 


88    THE  INOCULATION  OF  ANIMALS  AND  THEIR  STUDY. 

for.     If  it  is  invisible,  pressure  at  the  root  of  the  ear  will 
make  it  prominent. 

From  the  dorsal  surface  the  hypodermatic  needle  is  in- 
serted at  its  distal  extremity  and  the  material  slowly  injected. 
Care  should  be  taken  to  have  the  needle  penetrate  the  vein, 
and  the  first  few  drops  of  injected  liquid  will  show  whether 
it  does  so  or  not ;  for  if  the  needle  is  outside  the  vein  a  bulla 
will  immediately  be  found  at  the  site  of  injection.  A  little 
practice  renders  this  method  very  easy. 

4.  Inoculation  into  the  lymphatics  is  done  best  with  a  hypo- 
dermatic syringe  and  the  injection  is  made  into  the  testicles. 

5.  Intraperitoneal  inoculation  requires  much  care  and  the 
same  antiseptic  precautions  as  when  opening  the  peritoneum 
for  a  laparotomy.     The  skin,  cleanly  shaven  and  disinfected 
as  thoroughly  as  possible,  is  opened  in  the  linea  alba  midway 
between  the  sternum  and  pubis,  through  an  incision,  from  an 
inch  and  a  half  to  two  inches  long  and  penetrating  the  fascia. 
The  edges  of  the  wound   being  held  apart,   the  connective 
tissue  and   muscles  are  separated  with  a  pair  of  sterilized, 
blunt-pointed  scissors.       If   a  liquid  inoculating  material  is 
used,  it  may  now  be  introduced  with  a  sterile  hypodermatic 
needle  into  the  peritoneal  cavity,  avoiding  as  much  as  possi- 
ble the  wounding  of  the  intestines,  which  is  not  difficult.     If 
the  material  employed  is  solid,  the  peritoneum  is  opened  with 
scissors  and  the  solid  particle  introduced  into  the  cavity  by 
means  of  a  sterile  needle  or  forceps.     The  wound  is  care- 
fully sutured  and  closed  by  a  layer  of  collodion. 

6.  Intrapleural  inoculation  is  performed  much   more  rarely 
on  account  of  the  danger  of  wounding  the  lungs,  and  when 
used  the  same  precaution  must   be  taken  as  for  the  intra- 
peritoneal  method. 

7.  Inoculation  into  the  anterior  chamber  of  the  eye  is  per- 
formed occasionally  to  study  in  the  living  animal  the  changes 
produced  locally  by  bacteria.     With  a  sharp-pointed  bistoury 
an  incision  is  made  in  the  cornea,  at  its  sclerotic  attachment 
near  the  inner  canthus,  and   the    material   introduced   and 
applied  directly  upon  the  iris,  by  a  sterilized  needle  or  by 
means  of  a  small  forceps. 


THE  OBSERVATION  OF  THE  INOCULATED  ANIMAL.     89 

THE  OBSERVATION  OF  THE  INOCULATED  ANIMAL. 

1.  After  inoculation  animals  should  be  observed  carefully  and 
all  changes  in  their  condition  noted.    Their  temperature  should 
be  taken  several  times  a  day,  their  body  weight  recorded  every 
day  under    the   same   conditions ;    their  behavior  as  to  food 
noticed ;  the  state  of  their  fur  and  any  sign  of  paralysis  or  of 
convulsions  carefully  observed. 

2.  After  death,  the  autopsy  should  be  performed  as  soon  as 
practicable.     For   that  purpose  the  animal   should   be   laid 
upon  its  back  on  a  board,  its  four  legs  stretched  widely  apart 
and  attached  to  the  sides  of  the  board  by  strings,  or  nailed, 
and  the  nose  should  also  be  carefully  nailed  down.    By  means 
of  a  5  per  cent,  carbolic  acid  solution  the  skin  of  the.  body 
from  the  chin  to  the  pubis  should  be  carefully  sterilized  and 
all  hair  shaved  off.     The  place  of  inoculation  is  now  to  be 
carefully  examined  and  described. 

Examination  of  the  Abdominal  and  Pelvic  Contents. — After 
this  an  incision  is  made  in  the  skin  only,  and  that  carefully 
dissected  away  from  the  subcutaneous  tissue  and  hooked  back 
so  as  to  prevent  its  contaminating  the  underlying  tissues. 
After  this  by  means  of  a  metallic  spatula,  heated  to  redness, 
the  muscular  tissue  is  singed  all  along  the  line  of  the  next 
incision,  along  the  linea  alba  from  the  pubis  to  the  sternum, 
along  the  arches  of  the  rib  and  obliquely  from  the  sterno- 
clavicular  junctions  to  the  tip  of  the  last  two  ribs.  With  a 
pair  of  sterilized  blunt-pointed  scissors  the  peritoneal  cavity 
is  opened.  All  changes  within  it  are  carefully  noted,  cultures 
made  from  all  exudates,  or  inflammatory  products  apparent, 
bouillon,  agar,  and  blood-serum  tubes  being  used  for  that  pur- 
pose, and  cover-glasses  being  also  prepared  for  the  microscope. 
Then,  by  means  of  the  same  heated  spatula,  the  surfaces  of  the 
different  organs  are  thoroughly  singed,  and  with  a  spear-head, 
thick  sterilized  platinum  wire,  the  organ  is  penetrated  and 
cultures  made  from  the  small  pieces  of  organs  or  blood 
adhering  to  the  wire ;  bouillon,  agar,  and  blood-serum  being 
used,  and  cover-glasses  being  also  prepared. 

From  all  cultures  so  obtained  plate  cultures  should  be  made. 


90    THE  INOCULATION  OF  ANIMALS  AND   THEIR  STUDY. 

and   the  several  bacteria  therein   isolated,  and  pure  cultures 
made. 

After  complete  examination  of  the  abdominal  and  pelvic 
organs,  by  means  of  a  thick  pair  of  scissors  the  ribs  are  cut 
off  along  the  singed  lines,  the  sternum  turned  up,  and  exam- 
ination and  cultures  of  the  thoracic  organs  made  in  the  same 
way  as  for  the  abdominal  organs. 

3.  Alter  the  complete  autopsy  the  animal  should  be  inciner- 
ated, and  if  this  is  not  practicable,  it  should  be  kept  bathed 
at  least  for  two  hours  in  a  5  per  cent,  carbolic  acid  solution, 
and  finally  boxed  in  quicklime  before  burial. 

Cultures  from  the  human  body  at  autopsies  should  be  made 
as  described  for  lower  animals. 

4.  Cultures  from  secretions  of  living  animals  and  man  should 
be  made  immediately  upon  their  passage  and  must  be  always 
collected  in  sterilized  vessels.    The  examinations  and  the  cult- 
ures should  be  prepared  forthwith,  using  agar,  bouillon,  and 
serum  as  media. 


The  Roux-Nocard  Method  of  Culture  and  Observation. 

History. — Recently  observations  made  by  Roux  and  Nocard 
for  the  growth  of  microorganisms  in  culture  in  the  live 
animal  have  shown  that  a  number  of  minute  bacteria  are 
found  associated  with  certain  diseases,  notably  the  l^leuro- 
pneujnonia^pf  cattle.  These  microorganisms  require  a  much 
TngKer  power  of  tKe  microscope  than  that  generally  in  use  to 
bring  them  into  view,  a  magnification  of  2000  at  least  being 
necessary. 

Technic. — Small  collodion  flasks  are  made  thoroughly 
sterile,  filled  with  blood  from  the  suspected  animal,  and  then 
closed  with  sterile  collodion.  These  tubes  or  flasks  are  after- 
ward introduced  into  the  abdominal  cavity  of  live  rabbits  and 
guinea-pigs,  and  allowed  to  remain  for  a  few  days,  after  which 
they  are  taken  out  and  examined,  when  the  small  motile 
microorganisms  as  mentioned  above  will  be  discovered. 

Importance. — This  method  of  observation  and  cultivation 
in  the  live  animal  body  seems  to  open  a  large  field  for  the 


INFECTION  AND  IMMUNITY.  91 

future  investigation  of  such  diseases  as  scarlet  fever,  measles, 
smallpox,  rabies,  etc.,  which,  though  unquestionably  of 
microbic  origin,  have  so  far  failed  to  reveal  any  specific  germ 
for  their  causation. 

QUESTIONS. 

Why  are  animals  inoculated  ? 

What  different  methods  are  used  for  animal  inoculation  in  the  laboratory  ? 

Describe  the  subcutaneous  method.  The  intravenous  method.  The  intra- 
lymphatic  method.  The  intraperitoneal  method. 

What  instruments  are  used  to  inoculate  liquid  cultures  into  the  veins  of 
animals  ? 

What  should  be  observed  in  inoculated  animals? 

How  should  an  autopsy  be  made  in  the  case  of  an  animal  dead  after 
inoculation  V 

What  precautions  are  necessary  in  making  cultures  from  tissues  and  organs 
in  dead  animals  to  prevent  contamination  from  outside  ? 

How  should  secretions  of  animals  and  men  be  collected  for  bacterial  exam- 
ination ? 

What  form  of  culture  have  Eoux  and  Nocard  proposed  for  cultivation  of 
bacteria  ? 


CHAPTER  VII. 

INFECTION   AND   IMMUNITY. 

INFECTION. 

BACTERIA  which  produce  diseases  in  animals  and  man  are 
known  as  the  pathogenic  bacteria,  and  the  process  by  which 
disease  is  produced  is  called  infection. 

The  mode  of  communication  of  these  infections  to  man  or 
to  animals  is  not  fully  demonstrated.  The  following  explana- 
tions are  the  more  plausible. 

The  Theories  of  Infection.  (P>£ 

1.  The  rapid  multiplication  of  bacteria  in  the  blood  and 
organs  of  infected  animals  is  supposed  to  interfere  with  their 
bodily  functions,  and  so  cause  disease  and  death.  This  is  the 
so-called  mechanical  theory  of  infection,  and  finds  support  in 
such  diseases  as  anthrax,  when  in  fatal  cases  every  capillary 


92  INFECTION  AND  IMMUNITY. 

and  organ  of  the  animal  is  teeming  with  microorganisms, 
and  in  so-called  septicsemic  diseases  where  the  microorgan- 
isms may  be  found  in  greater  or  lesser  number  in  the  blood 
and  organs. 

II.  The  bacteria  secrete  or  contain  in  their  cell-bodies 
poisonous  substances  (toxins)  which  act  deleteriously  on  the 
animal  economy  through  its  own  molecules.  This,  the  chemi- 
cal theory,  is  the  accepted  one  of  to-day,  and  finds  its  ready 
explanation  in  nearly  all  infectious  diseases,  especially  in 
those  which,  like  diphtheria  and  tetanus,  may  be  superin- 
duced by  inoculations  of  cultures  from  which  the  bacteria 
have  been  eliminated  by  filtration.  The  so-called  toxaemic 
diseases  are  so  produced. 

The  Avenues  and  Factors  of  Infection. 

A.  Infection  of  the  animal  body  is  effected  by  one  of  three 
ways  : 

I.  Through  the  respiratory  tract. 

II.  Through  the  digestive  tract. 

III.  Through   the  wounded  or  unwounded   surface   of  the 
skin  or  mucous  membrane. 

B.  Conditions  and  Factors. — These  are  various  and  play  an 
important  part  in  infection.     Some  of  them  have  reference 
(1)  to  the  infecting  material,  or  chiefly  (2)  to  the  animal  ex- 
perimented upon.     To  the  first  class  belong  the  species  of 
bacteria,   the  quantity   of  infected  material   introduced,  the 
cultural  conditions  of  the  bacteria,  the  presence  or  absence 
of  the  so-called   mixed    infection   in  which  more  than  one 
species  is  taking  part,  the  method  of  its  introduction,  and, 
in  some  cases,  the  time  elapsed  since  the  infection  occurred. 
The  conditions  which  depend  upon  the  animal  are  the  follow- 
ing :  the  amount  of  natural  resistance  to  the  bacterial  poison, 
the  condition  of  health  of  the  animal. 

It  must  be  remembered  that  some  species  of  bacteria  are 
much  more  injurious  than  others  either  on  account  of  the 
rapidity  with  which  they  are  able  to  develop  in  the  human 
or  animal  economy,  or  on  account  of  the  large  quantity  of 


INFECTION.  93 

toxins  which  they  generate,  or  on  account  of  the  highly  poi- 
sonous property  of  these  toxins. 

1 .  The  quantity  of  bacteria  used  plays  an  important  part 
because  there  is  a  more  or  less  marked  natural  resistance  in 
the  animal   body  to  the  action  of  bacteria  or  their  poisons. 
When  these  are  introduced  in  small  quantity  only,  they  fail 
to  produce  any  effect,  and  it  requires  a  certain  definite  amount 
of  bacteria  to  produce  disease   in    the  animal  body.     This 
amount  varies  with  the  species  of  the  bacteria. 

2.  The  condition  of  the  bacterial  culture  when  introduced 
into  the  animal  body  is  an  important  factor  in  the  subsequent 
course  of  the  infection,  for  bacteria  under  different  conditions 
secrete  toxins  which  are  more  or  less  injurious,  and  the  same 
bacteria  grown  under  the  same  conditions  are  able  at  different 
times   to  produce  toxins  of  more  or  less  virulence.     When 
the  "condition  of  growth  or  the  environment  of  the  bacteria 
varies,  their  cultural  aspects  and  the  amount  of  toxins  they 
are  able  to  produce  vary  also.     So  much  so  is  this  the  case 
that  bacteria   are  grown    under   peculiarly  disadvantageous 
surroundings — high  temperature,  or  the  addition  of  a  small 
proportion  of  antiseptics  to  their  cultural  fluid  so  as  to  pro- 
duce bacteria  of  less  virulence — in  other  words,  to  attenuate 
them. 

Methods  of  Attenuation. — Bacteria  from  young  liquid  cult- 
ures are  known  to  be  more  virulent  than  those  from  older 
cultures.  Again,  cultures  are  made  through  the  body  of 
resisting  animals  so  as  to  diminish  the  virulence  of  the  cult- 
ures. Or,  again,  the  cultures  are  passed  through  artificial 
media  for  a  number  of  generations  to  diminish  their  virulence. 
The  converse  of  this  happens  also,  and  bacteria  grown  or 
passed  through  the  bodies  of  susceptible  animals  acquire  more 
and  more  virulence. 

3.  The  method  of  introduction  of  the  bacteria  contributes 
considerably  to  the  degree  of  infection  from   the  fact  that 
nearly  all  bacteria  have  certain  affinities  for  different  tissues 
of  the  body  where  they  exert  their  most  baneful  influence, 
and  the  nearer  akin  to  those  tissues  is  the  place  of  the  intro- 
duction in  the  body  the  more  rapidly  and  energetically  is  the 


94  INFECTION  AND  IMMUNITY. 

bacterial  influence  felt.  Again,  the  different  secretions  of 
the  body  have  more  or  less  germicidal  effect,  so  that  bacteria, 
as  a  rule,  are  more  potent  in  their  effect  when  introduced 
directly  into  the  circulation. 

4.  The  association  of  bacteria  among  themselves  has  occa- 
sionally the  power  of  increasing  the  toxic  effects  of  the  inocu- 
lated germs,  sometimes  the  two  germs  acting  simultaneously 
on  the  animal  body  and  producing  what  is  known  as  "  double," 
"mixed,"  or  "associated"  infection.     At  other  times,  some  of 
the  germs,  though   not  pathogenic,  are  able  to  destroy  the 
resistance  of  the  body  to  the  action  of  other  toxic  germs,  as, 
for   instance,  the    injection    of   tetanus   bacillus   with    some 
ordinary  saprophyte  is  capable  of  producing  symptoms  when 
the  introduction  of  the  tetanus  germs  alone  would  utterly 
fail. 

Occasionally  a  beneficial  association  of  germs  may  be  ob- 
served, the  presence  of  the  secretion  of  some  bacteria  being 
prejudicial  to  the  growth  of  other  bacteria  or  neutralizing 
their  toxins. 

5.  The  condition  of  the  human  or  animal  body  as  to  per- 
fect health,  as  has  already  been  remarked,  offers  more  or  less 
resistance  to  the  bacterial  poison.     When,  however,  the  gen- 
eral health  is  below  par  this  resistance  is  diminished  and  the 
animal  is  much  more  susceptible  to  the  action  of  the  germs. 

6.  The  time  elapsed  since  the  infection  is  often  of  great 
moment.     In  some  cases  germs  will  lurk  in  an  organ  for  a 
long   time,   after   which,  through    circumstances    very    little 
understood,  they  will  suddenly  and  violently  begin  to  cause 
symptoms  and  often  death.     Diseases  of  the  appendix  and 
gall-bladder  in   man  are  among  the  more  familiar  examples 
of  this  phenomenon. 

IMMUNITY  AND  ITS  VARIETIES. 

Resistance  to  the  action  of  pathogenic  bacteria  is  called 
immunity,  and  is  either  natural  or  acquired. 

I.  Natural  immunity  is  present  in  all  such  cases  where,  for 
instance,  some  species  of  animals  can  not  be  affected  by  cer- 


IMMUNITY  AND  ITS  VARIETIES.  95 

tain  bacteria  or  their  toxins,  which  are  injurious  to  other 
species,  or,  as  occasionally  happens,  when  some  individuals 
in  a  susceptible  species  are  refractive. 

II.     Acquired  immunity  is  manifested  when  a  susceptible 

animal  is  protected  from  the  further  noxious  influences  of 

bacteria  either  from  the  fact  of  having  suffered  an  attack  of 

the  disease  caused  by  the  bacteria,  or  when  it  has  been  made 

V, artificially  insusceptible. 

Examples  of  Natural  Immunity. — Rats  can  not  be  success- 
fully inoculated  with  the  anthrax  bacillus,  though  other 
rodents  are  very  susceptible.  Again,  pigeons  are  not  sus- 
ceptible but  are  immune  to  the  anthrax  bacillus.  The  expla- 
nation of  this  natural  immunity  is  not  easily  given.  It  is 
supposed  in  some  cases  to  be  due  to  the  mode  of  living  of 
the  immune  animal,  or  to  some  condition  of  its  secretions,  or 
to  some  substances  found  in  its  blood  and  tissues  which  are 

\j,  able  to  destroy  bacterial    life  or  to  neutralize   their  toxins. 

*>T  These  substances  are  called  alexins. 

Examples  of  Acquired  Immunity.— This  may  be  due,  as  just 
mentioned,  to  a  previous  attack  of  disease,  and  when  due  to 
this  it  lasts  in  the  majority  of  instances  during  the  life  of 
the  animal.  In  other  cases  acquired  immunity  can  be  arti- 
ficially induced  in  animals,  and  according  to  the  methods 
used  for  its  production  is  said  to  be  active  or  passive. 

1.  Active  acquired  immunity  is  produced  by  the  action  of 
living  germs  or  their  toxins  introduced  into  the  animal. 

2.  Passive  acquired  immunity  is  obtained  by  a  direct  trans- 
ference of  an  immunizing  substance  from  an  immune  animal 
to  a  susceptible  one.     Active  immunity  takes  some  time  to 
develop,  but,  as  a  rule,  lasts  longer  than  passive  immunity, 
which  is  immediately  established. 

The  Methods  of  Producing  Immunity. 

I.  Inoculation,  or  the  introduction  of  small  quantities  of  live 
bacteria,  so  as  to  produce  a  mild  attack  of  the  disease.  This 
method  is  dangerous  from  the  fact  that  it  is  hard  to  ascertain 
how  small  a  quantity  of  bacteria  may  be  introduced  without 


96  INFECTION  AND  IMMUNITY. 

its  being  prejudicial  to  life,  and  from  the  danger  of  spread- 
ing the  infection. 

II.  Vaccination,  or  the  introduction  of  attenuated  bacteria, 
which  attenuation  is  obtained  either  by  submitting  the  bacteria 
to  a  higher  degree  of  heat  during  their  cultivation  or  by  add- 
ing a  small  proportion  of  an  antiseptic  to  their  culture-media, 
or  by  using  bacteria  which  have  grown  for  a  long  period  of 
time  in   artificial   media,  or  by  using  bacteria  which  have 
grown  in  the  bodies  of  natural  immunes. 

III.  Intoxication,  or  the  introduction  of  the  toxins  of  the 
bacteria  in  small  broken  but  frequently  repeated  doses,  or  in 
cases  where  the  toxic  effect  of  bacteria  is  due  to  substances 
contained  in  the  cell-body  itself  by  the  injection  of  the  dead 
bacilli.     This  is  the  method  used  for  the  production  of  the 
diphtheria  and  tetanus  antitoxins. 

IV.  Antitoxins,   or  the  introduction  of   bacterial  products 
of  any  one  of  the  first  three  processes  into  other  animals, 
these  substances,   known  by  the  name  of  antitoxins,  being 
able  to  confer  immunity  to  susceptible  animals. 

V.  By  the  inoculation  of  an  emulsion  of  tissues,  consisting 
in  the  introduction  into  the  animal  of  the  emulsion  of  certain 
tissues  which  are  known  to  be  the  tissues  susceptible  to  the 
action  of  the  bacterial  poison. 

VI.  By  introducing  into  the  animal  inert  particles,  such  as 
carmine,  mixed  with  the  bacteria. 

Forced  immunization  of  animals  consists  in  introducing 
gradually  and  in  increasing  doses  bacterial  toxins  in  sufficient 
quantity  to  produce  a  reaction,  but  in  quantity  too  small  to 
produce  deleterious  effects.  In  this  way  it  has  been  found 
that  animals  immunized  produce  substances  in  their  tissue- 
fluids  which  when  inoculated  in  susceptible  animals  serve  to 
protect  them  against  the  deleterious  action  of  those  bacteria 
or  their  toxins. 

The  Antitoxic  and  Antimicrobic  Blood-Serums. 

The  blood-serum  of  animals  used  for  the  purpose  of  pro- 
tecting others  is  said  to  be  antitoxic,  when  it  has  been  obtained 


IMMUNITY  AND  ITS   VARIETIES.  97 

by  the  action  of  the  toxins  of  the  bacteria  on  the  animal ; 
and  to  be  antimicrobic,  when  it  has  been  obtained  by  means 
of  the  action  of  virulent  or  attenuated  cultures  on  those 
animals. 

Uses. — Antitoxic  serum  is  employed  chiefly  in  the  toxic 
diseases,  such  as  diphtheria,  tetanus,  etc.,  and  antimicrobic 
serum  is  used  particularly  in  the  invasive  diseases,  such  as 
plague,  typhoid  fever,  cholera,  etc. 

Theories  in  Explanation. — A  number  have  been  suggested. 
Some  believe  that  the  antitoxin  is  a  chemically  changed  toxin  ; 
others  claim  that  it  is  a  sort  of  enzyme  produced  by  the  toxin  ; 
others  again  state  that  it  is  the  product  of  the  cytic  activity 
developed  by  the  toxin;  again  others  consider  that  it  acts  as 
a  sort  of  combining  ferment  in  the  same  manner  as  those  fer- 
ments which  favor  coagulation  of  the  fibrin  in  the  blood. 

The  Theories  of  Immunity. 

How  these  substances  act  so  as  to  produce  immunity  in  ani- 
mals is  a  subject  that  has  occupied  investigators  considerably  in 
recent  years. 

I.  The  abstraction  theory  (Pasteur's)  is  to-day  only  of  his- 
torical interest.     It  was  believed  to  be  due  to  the  fact  that 
the  pabulum  necessary  for  the  life  of  the  specific  bacteria  had 
been  consumed,  and  that   these  bacteria  could  no  longer  live 
in  the  animal. 

II.  The  retention  theory  (Chauveau's),  in  which  it  was  sup- 
posed that  microorganisms  left  in  the  system  certain  substances 
which  were  antagonistic  to  their  further  growth,  is  still  worthy 
to-day  of  some  consideration. 

III.  The  theory  of  phagocytosis  (Metchnikoff's),  by  which 
immunity  was  supposed  to  be  due  to  the  action  of  the  white 
blood-corpuscles,  which    have   the   power  of  absorbing  and 
destroying   bacteria,    is    not   tenable   to-day   in   its  original 
entirety.     That   the   leucocytes   play  a  certain   part  in  the 
immunizing  process  cannot   be  denied,   but  the  phagocytic 
property  is  more  probably  due  to  the  fact  that  the  animal  is 
immune  than  the  cause  of  the  immunization. 

7— M.  B. 


98  INFECTION  AND  IMMUNITY. 

Immunity  is,  in  general  terms,  certainly  produced  by  certain 
secretions  formed  in  the  animal's  body,  and  secreted  by  it  to 
protect  itself  from  the  attack  of  the  invading  bacteria,  and  dis- 
tributed in  all  the  tissues,  but  found  especially  in  the  serum  of 
the  blood. 

IV.  The  chain-theory  (Ehrlich's)  claims  that  this  immuniz- 
ing substance  is  developed  on  account  of  the  fact  that  the  poi- 
sonous substances  introduced  by  the  microbes  or  the  secretions 
of  the  microbes  in  the  animal  body  combine  with  certain 
elements  of  the  tissue  and  destroy  them,  subtracting  them 
from  other  elements  with  which  they  were  naturally  in  com- 
bination ;  this  stimulates  the  natural  resistance  of  the  tissues 
and  causes  an  increased  production  of  the  substances  attacked 
by  the  bacteria  in  such  a  way  that  an  overproduction  results, 
and  this  makes  the  animal  more  resistant  to  the  further  intro- 
duction of  the  poison.  This  is  certainly  the  most  plausible 
explanation  of  immunity  offered  to  this  day. 

A  passing  remark,  however,  may  only  be  offered  on  this 
subject,  and  those  who  are  interested  must  be  directed  to  con- 
sult larger  works,  in  which  these  views  are  explained  at 
length. 

QUESTIONS. 

What  is  infection  ? 

How  are  bacteria  called  which  produce  disease  in  animals? 

How  is  the  action  of  pathogenic  bacteria  on  the  animal  body  explained  ? 

When  is  a  disease  said  to  be  septicaemic  ?    When  is  it  toxjemic  ? 

Name  the  three  modes  by  which  the  animal  body  may  be  infected. 

What  conditions  favor  infection  ? 

What  conditions  in  the  infecting  material  increase  its  power? 

What  conditions  in  the  animal  increase  the  rapidity  of  infection  ? 

What  part  does  the  quantity  of  bacteria  introduced  in  the  inoculation  play 
in  the  infection? 

What  is  meant  by  attenuation  ? 

What  conditions  of  the  cultures  make  the  bacteria  more  virulent? 

What  is  the  effect  of  passing  for  a  number  of  generations  pathogenic  bac- 
teria through  artificial  media  ? 

What  part  does  the  mode  of  introduction  of  the  bacteria  in  the  animal  body 
play  in  infection? 

What  is  meant  by  double  infection  ? 

What  is  immunity  ? 

What  is  meant  by  natural  immunity  ? 

What  is  meant  by  acquired  immunity  ? 

Give  some  examples  of  natural  immunity  ? 

What  produces  acquired  immunity  in  animals? 


THE  PATHOGENIC  BACTERIA.  99 

What  are  active  and  passive  immunity? 
What  artificial  methods  are  used  to  produce  immunity? 
What  is  meant  by  inoculation  ?     Vaccination  ?     Intoxication  ? 
How  does  tissue  suspension  produce  immunity  ? 

What  influence  does  the  injection  of  inert  particles  have  upon  immunity? 
What  is  meant  by  forced  immunity  ? 

What  is  meant  by  an  antitoxic  serum  ?     By  an  antimicrobic  serum  ? 
What  classes  of  disease  are  protected  against  by  antitoxic  serum?    What 
by  antimicrobic  serum  ? 

How  is  this  anti-action  explained? 
What  is  the  theory  of  abstraction  ? 
What  is  meant  by  the  retention  theory  ? 
What  is  the  theory  of  Metchnikoff ? 
What  is  Ehrlich's  chain-theory? 


CHAPTER  VIII. 

THE  PATHOGENIC  BACTERIA. 

THE  PYOGENIC  MICROCOCCI  AND  ALLIED  BACILLI. 

THE  most  commonly  found  bacteria  in  pus  are  cocci  (pyo- 
cocci).     A  few  are  bacilli.     The  list  includes: 

1.  The  Staphylococcus  pyogcnes  aureus,  albus,  and  citreus ; 
the   Staphylococcus    cereus   albus,    the   Staphylococcus   cereus 
aureus  (Passet) ;  the  Staphylococcus  cereus  flavus  (Passet). 

2.  The    Micrococcus  pyogenes   tennis    (Rosenbach).      The 
Micrococcus    tetragenus   is  sometimes   found  associated  with 
the  foregoing  two  varieties  in  abscesses  or  in  pus  cavities, 
and  are  also  able  to  produce  abscesses  at  the  place  of  injec- 
tion in  animals. 

3.  The  Streptococcus  pyogenes  is  found  associated  with  the 
staphylococci   in   purulent  accumulations,  and  is  sometimes 
itself  responsible  for  pus-production  in  the  body. 

4.  The  gonococcus  is  the  cause  of  specific  suppuration  of 
the  urethra  and  often  elsewhere  in  the  body 

5.  The  pneumococcus   is    often   found    in   abscesses  which 
occur  in  the  course  of;  the;  disease  in- pneumonic  patients. 

6.  7,  and  8.  The  Baeiliux  />//o";/" //.""«*.  iyphosrs,  and  tuber- 
culosis are  sometimes  the  cause  of  pus-prod'uctioir,  'as -pure 


100  THE  PATHOGENIC  BACTERIA. 

cultures  of  these  organisms  have  been  found  in  some  cases 
of  abscesses  during  the  respective  infections. 

Nearly  all  pyogenic  organisms  are  facultative  anaerobics. 

THE  INDIVIDUAL  FEATURES  OF  THE  PYOGENIC 
BACTERIA. 

I.  Staphylococcus  Pyogenes  Aureus. 

The  Staphylococcus  pyogenes  aureus,  by  far  the  most  fre- 
quent pus  organism,  is  found  a.  in  health  on  the  surface  of  the 
skin,  also  of  the  mucous  membranes  in  the  digestive  tube, 
and  upper  part  of  the  respiratory  tract,  and  b.  in  pathological 
conditions  in  pus  irrespective  of  its  localization,  either  alone 
or  in  association  with  the  other  pyogenic  staphylococci,  also 

FIG.  47. 


Preparation  from  pus,  showing  pus-cells,  A,  and  staphylococci,  C.    (Abbott.) 

in  the  blood  in  cases  of  general  infection,  and  a  number 
of  cases  of  extensive  suppurating  lesions,  abscesses,  suppu- 
rating tumors,  furuncles,  etc.;  and  c.  outside  of  the  human 
body  in  the  air,  in  dust,  and  occasionally  in  water. 

Morphology. — The  Staphylococcus  pyogenes  aureus  is  a 
small  ,  rounded,  celL. having  a«  diameter  of  0.9  to  1.2  mikrons, 
found  "either',  singly  ;c"r^;in  fee^lar,  groups  or  masses  resem- 
bling a  bunch  of1  grapes,  hence  its  name.  Sometimes  it  is  seen 


INDIVIDUAL  FEATURES  OF  PYOGENIG  BACTERIA.   101 

in  pairs,  as  a  diplococcus.  Its  appearance  in  pus  as  well  as 
in  culture-media  is  the  same  in  general  as  is  seen  in  Fig.  47. 

Principal  Biologic  Characters. — The  Staphylococcus  pyogenes 
aureus  is  a  facultative  anaerobic.  It  clouds  bouillon  in  twenty- 
four  hours  at  37°  C.,  and  shows  from  the  second  day  a  yel- 
lowish precipitate,  which  gradually  increases  in  color  and  at 
the  bottom  of  the  tube  appears  of  a  golden  yellow.  It  lique- 
fies gelatin.  Stab-cultures  on  this  media  at  20°  C.  on  the 
second  or  third  day  have  the  appearance  of  a  funnel,  at  the 
bottom  of  which  is  an  orange-yellow  deposit.  At  the  end 
of  three  days  the  gelatin  in  the  tube  is  completely  liquefied. 
On  gelatin  plates  colonies  of  a  dark-yellowish  color  are 
observed  with  a  centre  of  more  or  less  intense  orange  color. 

On  agar-agar  the  colonies  appear  small,  regularly  spherical, 
and  of  an  orange-yellow.  Plates  made  from  this  medium 
have  the  same  characteristics  as  on  gelatin,  being  more  or 
less  pigmented  yellow.  It  does  not  liquefy  agar.  The  cult- 
ures on  blood-serum  have  the  same  characteristics  as  on  agar. 
The  Staphylococcus  pyogenes  aureus  stains  with  all  the  anilin 
dyes,  and  also  by  Gram's  method. 

Pathogenesis. — When  inoculated  into  the  blood  of  an  animal, 
the  Staphylococcus  aureus  rapidly  causes  a  fatal  septicaemia. 
Rabbits  and  guinea-pigs  die,  as  a  rule,  in  twenty-four  to 
forty-eight  hours  after  inoculation,  and  the  organisms  may  be 
found  generally  disseminated  in  the  blood-capillaries  of  the 
organs,  and  are  also  found  in  the  blood  taken  from  the  heart. 

Inoculations  into  the  peritoneal  cavity  cause  a  purulent 
peritonitis  of  a  virulent  character,  generally  ending  in  death 
of  the  animal.  Injected  under  the  skin,  this  organism  pro- 
duces localized  abscesses. 

II.   Staphylococcus  Pyogenes  Albus. 

The  Staphylococcus  pyogenes  albus,  like  its  companion  the 
aureus,  exists  as  a  saprophyte  :  a.  on  the  surface  of  the  skin 
in  man,  and  b.  in  association  with  the  aureus  in  abscesses 
and  superficial  phlegmons. 

Although  clinicians  are  in  the  habit  of  considering  it  as  an 


102  THE  PATHOGENIC  BACTERIA. 

achromogenic  variety  of  the  preceding,  it  is,  however,  some- 
what less  pathogenic.  Its  morphological  characters  are  the 
same  as  those  of  the  aureus,  with  the  exception  that  it  does 
not  form  pigment  and  its  colonies  are  of  a  milk-white  color. 

III.  Staphylococcus   Citreus. 

The  Staphylococcus  pyogenes  citreus  is  of  identical  mor- 
phology with  the  two  preceding  varieties,  with  the  exceptions 
that  its  growth  is  of  a  lemon-yellow  color  and  that  it  liquefies 
gelatin  more  slowly.  It  is  found  in  association  with  the  Sta- 
phylococcus aureus  and  albus  in  pus  of  acute  abscesses,  espe- 
cially in  the  liver. 

IV.  Streptococcus  Pyogenes. 

The  Streptococcus  pyogenes  is  found :  a.  in  the  lymphatics 
of  the  skin  in  patients  suffering  from  erysipelas,  b.  in  pus, 
c.  in  the  false  membranes  in  cases  of  diphtheria,  d.  in  surgi- 
cal and  e.  obstetrical  complications  of  erysipelas,  and  /.  as  a 
frequent  causative  agent  of  puerperal  septicaemia  and  of  many 
surgically  common  infections  (Fig.  48  and  Plate  I.). 

FIG.  48. 


Streptococcus  pyogenes.    (Abbott.) 

Morphology. — The  streptococcus  is  a  micrococcus  varying 
in  size  from  1  to  4  mikrons  in  diameter,  spherical  in  shape 
and  arranged  as  a  chain  of  variable  length.  When  grown  in 
liquid  media,  this  chain  consists  of  from  30  to  40  elements, 
but  in  solid  media  a  chain  usually  consists  of  from  7  to  10 
cocci,  In  young  cultures  the  diameters  of  all  the  cocci  of  the 


PLATE  I. 


Streptococcus  Pyogenes  in  Pus.     (Abbott.) 


INDIVIDUAL  FEATURES  OF  PYOGENIC  BACTERIA.   103 

chain  are  equal ;  in  older  cultures  they  vary  very  much  even 
in  the  same  chain. 

Biologic  Characters. — The  streptococcus  is  aerobic  and  fac- 
ultative anaerobic.  At  37°  C.  it  clouds  bouillon  in  twenty- 
four  hours,  and  this  becomes  again  clear  at  the  end  of  three 
or  four  days,  when  small  spherical  bodies  may  be  seen  at  the 
bottom  of  the  tube.  The  bouillon  becomes  acid.  On  gelatin 
it  forms  small  spherical  opaque  colonies  about  the  size  of  a 
pin-head,  which  cease  to  increase  after  the  third  or  fourth 
day.  It  does  not  liquefy  gelatin.  On  agar-agar  also  it  forms 
spherical  colonies  of  the  size  of  a  pin-head,  semitransparent 
and  of  a  grayish- white  appearance,  shaped  somewhat  like  a 
bead.  It  does  not  develop  on  potato.  The  streptococcus  does 
not  live  longer  than  three  weeks  in  cultures.  It  stains  by  the 
Gram  method,  and  also  by  the  other  anilin  dyes. 

Pathogenesis. — Intravenous  inoculations  in  animals  produce 
variable  effects.  The  germ  usually  kills  the  animal,  causing 
a  rapid  general  septicaemia  ;  at  other  times  the  animal  reacts 
only  slightly.  Subcutaneously  it  causes  erysipelas  and  the 
formation  of  abscesses.  All  laboratory  animals  are  susceptible 
to  infection  by  means  of  the  streptococcus  pyogenes. 

V.  The  Micrococcus  Cereus  Albus. 


VI.  The  Micrococcus  Cereus  Flavus. 

These  were  found  in  pus  by  Passet  associated  with  other 
organisms.  Their  pathogenesis  has  not  been  fully  established. 
They  differ  from  the  other  groups  of  cocci  just  described  by 
the  shiny,  waxy  appearance  of  their  growth. 

VII.  The  Micrococcus  Pyogenes  Tenuis. 

This  was  found  in  pus  by  Rosenbach,  is  very  irregular  in 
size  and  somewhat  larger  than  the  Staphylococcus  albus.  On 
agar-agar  its  biology  presents  a  thin  opaque  streak  along  the 
line  of  inoculation,  resembling  a  thin  layer  of  varnish.  Its 
pathogenic  properties  have  never  been  fully  determined. 


104  THE  PATHOGENIC  BACTERIA. 

VIII.  Micrococcus  Tetragenus. 

The  Micrococcus  tetragenus  was  obtained  by  Koch  a.  from 
cavities  of  tuberculous  lungs,  b.  in  the  sputum  of  phthisical 
patients  in  the  last  stages  of  the  disease,  c.  in  the  pus  of 
buccal  and  d.  ocular  abscesses.  It  has  been  found  by  Morinier 
e.  in  the  normal  saliva  and  /.  even  in  the  saliva  of  newborn 
babes. 

Morphology. — A  micrococcus  with  a  diameter  of  about  1 
mikron,  formed  in  groups  of  8  (tetrads)  and  enveloped  by  a 
transparent  gelatinous  substance. 

Principal  Biologic  Properties. — It  is  a  facultative  anaerobic. 
On  agar  it  forms  thick  granular  spherical  colonies  of  a  white 
or  grayish  color.  It  does  not  liquefy  gelatin.  It  stains  with 
all  the  anilin  dyes  and  readily  by  Gram's  method. 

Pathogenesis. — When  inoculated  into  guinea-pigs  subcutane- 
ously,  the  animals  die  rapidly  and  abscesses  are  formed  at  the 
point  of  inoculation.  The  micrococcus  at  the  autopsy  may 
be  found  in  all  the  organs  and  in  the  blood  taken  from  the 
heart. 

GONORRHCEA. 
IX.  Micrococcus  Gonorrhoeas  (Gonococcus). 

Discovered  by  Neisser  in  1879,  the  gonococcus  causes  the 
specific  suppuration  of  gonorrhoea. 

Pathogenesis. — This  micrococcus,  or  diplococcus,  as  it  is 
generally  called,  has  a  special  affinity  for  the  urethral  mucous 
membrane,  finding  lodgement  in  the  epithelial  cells  lining 
this  canal.  It  sometimes  causes  inflammation  with  or  with- 
out suppuration  in  other  parts  of  the  human  body,  such  as 
the  conjunctiva,  appendages  of  the  uterus,  in  the  peritoneum 
and  articulations.  Cutaneous  and  muscular  abscesses  have 
occasionally  been  found  to  be  caused  by  the  gonococcus. 

Morphology. — These  micrococci  are  usually  found  united  in 
pairs  presenting  the  appearance  of  grains  of  coffee,  the  two 
opposing  sides  being  generally  flattened  or  concave.  In 
stained  preparations  the  flattened  surfaces  are  separated  by 
an  unstained  interspace.  The  gonococci  are  found  free  in 


QONOREHCEA.  105 

the  pus,  but  more  often  as  small  masses  in  the  pus  or  epithe- 
lial cells.  This  serves  partly  to  distinguish  them  from  other 
pus  cocci  (Fig.  49). 

Principal  Biologic  Characters. — It  is  aerobic,  but  is  very 
difficult  to  cultivate  outside  the  human  body.  A  number  of 
investigators  have  succeeded  in  cultivating  it  on  human  blood- 
serum  obtained  from  the  placenta  of  a  recently  delivered 
woman  ;  others  have  been  successful  with  ascitic  fluid  and 
with  the  fluid  of  hydrocele.  The  cultures  grow  at  a  tempera- 
ture of  between  30°  and  35°  C.  Finger  has  succeeded  in 
cultivating  it  in  sterile  acid  urine  with  0.5  per  cent,  of  peptone. 

FIG.  49. 


I  «   «..<„ 

'•  *^m. 


s  $m 


7; 

Pus  of  gonorrhoea,  showing  diplococci  in  the  bodies  of  the  pus-cells.    (Abbott.) 

The  gonococcus  will  not  grow  on  gelatin,  agar-agar,  potato, 
or  in  bouillon. 

It  stains  with  the  basic  anilin  dyes,  especially  with  gentian- 
violet.  It  does  not  stain  by  the  Gram  method.  This  is  a 
valuable  point  to  differentiate  it  from  the  pus  cocci,  which 
all  stain  by  the  Gram  method. 

Pathogenesis. — Toure  succeeded  in  causing  urethritis  in  dogs 
by  injecting  into  their  urethras  cultures  in  acid  media.  Finger 
and  (J-ohm  have  caused  acute  urethritis,  which  rapidly  disap- 
peared, by  intra-articular  injections  of  cultures  into  dogs  and 
rabbits.  Pus  containing  the  gonococci  when  inoculated  into 


106  THE  PATHOGENIC  BACTERIA. 

man  have  reproduced  the  .disease  in  many  instances.  Pus 
cultures  of  the  gonococci  have  also  given  positive  results  in 
many  cases :  Wertheim,  5  times  in  5  cases ;  Bockardt,  6 
times  in  10  cases ;  Finger,  3  times  in  14  cases.  Subcutaneous 
injections  of  the  culture  produce  considerable  tumefaction  and 
redness  at  the  point  of  inoculation,  but  no  abscess-formation. 

X.  Bacillus  Pyocyaneus. 

The  Bacillus  pyocyaneus  is  found  frequently  in  suppurating 
wounds,  especially  in  burns.  It  colors  the  pus  green  and  the 
dressings  a  bluish-green,  without  showing  any  color-influence 
on  the  local  condition  of  the  wound. 

Pathogenesis. — It  exists  in  pus  associated  with  other  micro- 
organisniSj  and  is  considered  an  inoffensive  saprophyte  in 
most  cases.  It  may,  however,  under  certain  conditions 
become  pathogenic. 

Morphology. — It  is  a  delicate  rod  with  rounded  or  pointed 
ends. 

Biologic  Characters. — It  is  aerobic  and  grows  readily  on  all 
artificial  media  and  imparts  to  them  a  bright-green  color.  It 
liquefies  gelatin  and  stains  readily  with  all  anilin  dyes. 

XI.  Bacillus  Pyogenes  Fcetidus. 

This  organism  was  first  obtained  by  Passet  from  suppurat- 
ing surfaces  in  the  vicinity  of  the  lower  bowel. 

Morphology. — It  is  a  short  bacillus  with  rounded  ends, 
usually  found  in  pairs  or  in  short  chains. 

Biology. — It  is  an  aerobic,  motile,  and  grows  on  all  media. 
Stains  with  all  the  anilin  dyes.  The  cultures  are  noted  on 
account  of  the  disagreeable  putrefactive  odor  which  they  emit. 
It  derives  its  name  from  this  feature. 

XII.  Pneumococcus  or  Pneumobacillus. 

Friedlaender  discovered  this  organism.  It  is  sometimes 
found :  a.  in  pus  associated  with  other  organisms  and  b.  in 
cases  of  pneumonia  as  the  sole  factor  of  the  disease  and  ite 


GONORRHCEA.  107 

secondary  abscesses.     The  pus  produced  by  it  is  thick,  and 
creamy  white  in  color. 

Pathogenesis. — It  frequently  causes  suppuration  in  the 
serous  membranes — pleura,  peritoneum,  pericardium,  and 
lungs.  It  has  also  on  some  occasions  caused  suppuration 
in  the  viscera  and  in  the  subcutaneous  and  deep  cellular 
tissue. 

XIII.  Bacillus  Coli  Communis. 
XIV.  Bacillus  Typhosus. 

XV.  Bacillus  Tuberculosis. 

These  three  organisms  are  sometimes  found  associated  with 
pus-formation,  and  have  been  thought  to  be  occasionally  the 
chief  suppurative  agents.  The  discussion  of  this  subject, 
however,  will  be  properly  taken  up  under  the  head  of  the 
description  of  these  bacilli. 

QUESTIONS. 

What  are  the  pyococci? 

Describe  the  Staphylococcus  pyogenes  aureus.  How  does  it  act  on  bouillon, 
on  gelatin,  on  agar? 

Where  is  this  organism  found  in  the  human  body  ?  Where  outside  of  the 
human  body? 

What  is  the  effect  on  animals  of  intravenous  injections  of  this  organism? 
What  of  subcutaneous  inoculation  ? 

In  what  respect  does  the  Staphylococcus  pyogenes  albus  differ  from  the 
aureus?  The  Staphylococcus  pyogenes  citreusf 

Describe  the  streptococcus  pyogenes.  Where  is  it  found?  Of  how  many 
elements  are  its  chains  formed?  What  is  the  effect  of  intravenous,  intni- 
peritoneal,  and  subcutaneous  inoculations? 

Where  were  the  MicrococcMS  cereus  albus  and  flavus  found,  and  by  whom? 

What  are  the  characteristics  of  the  Micrococcus  tetragenns  f 

What  is  the  gonococcus?  Where  is  it  found  ?  How  is  it  recognized  under 
the  microscope  ? 

What  media  are  best  suited  for  its  growth?  How  is  it  differentiated  from 
other  pus  cocci  ? 

What  other  bacteria  cause  suppuration  or  are  found  in  pure  cultures  in 
abscesses. 


108  THE  PATHOGENIC  MICROCOCCL 


CHAPTER   IX. 

THE  OTHER  PATHOGENIC  MICROCOCCI  AND  ALLIED 
BACILLI— MICROCOCCUS  PNEUMONIA,  EPIDEMIC 
CEREBROSPINAL  MENINGITIS,  AND  MALTA  FEVER. 

PNEUMONIA. 

I.  Micrococcus  Pneumonias  Crouposae  (Diplococcus 
Pneumoniae ;  Micrococcus  Pasteuri ;  Micrococcus 
of  Sputum  Septicaemia). 

History. — The  Micrococcus  pneumonice  crouposce  was  discov- 
ered in  September,  1880,  by  Sternberg,  in  the  blood  of  rabbits 
which  he  had  inoculated  subcutaneously  with  his  own  saliva; 
also  by  Pasteur,  in  December,  1880,  in  the  saliva  of  a  child 
who  had  died  of  pneumonia  in  a  Paris  hospital.  This  was 
confirmed  and  studied  by  Fraenkel,  Weichselbaum,  and  others. 

It  is  found:  a.  in  the  saliva  of  about  50  per  cent,  of  healthy 
individuals,  6.  in  the  rusty  sputum  of  pneumonic  patients 
and  in  the  fibrinous  exudation  of  75  per  cent,  of  the  cases  of 
pneumonia,  c.  in  a  large  number  of  cases  of  meningitis  com- 
plicating pneumonia  or  associated  with  pneumonia,  d.  occa- 
sionally where  no  pneumonia  exists,  e.  also  in  abscesses. 

Morphology. — Micrococcus  pneumonice  is  a  small  oval  coccus 
appearing  alone  or  united  in  pairs,  occasionally  forming  chains 
with  four  or  five  elements  resembling  streptococci.  In  the 
animal  body  it  is  generally  oval  and  double,  as  a  diplococcus, 
surrounded  by  a  capsule  (Fig.  50). 

In  solid  media  it  grows  as  a  micrococcus,  a  diplococcus,  or 
as  a  chain  like  the  streptococcus  with  scarcely  more  than  four 
or  five  elements.  In  liquid  media  the  cells  are  more  nearly 
round,  and  the  chains  contain  sometimes  as  many  as  eight  or 
ten  elements  (Fig.  51). 

It  stains  by  the  anilin  dyes,  and  also  by  Gram's  method. 

Biologic  Characters. — The  Micrococcus  pneumonice,  is  aerobic 
and  facultative  anaerobic,  Like  most  cocci  it  is  non-motile, 


PNEUMONIA. 

FIG.  50. 


109 


Diplococcus  of  pneumonia  from  blood,  with  surrounding  capsule.    (Park.) 

and  therefore  has  no  flagella.  It  grows  on  all  culture-media, 
very  little  at  a  temperature  below  24°  C.,  best  at  a  tempera- 
ture of  37°  C.  At  a  temperature  above  42°  C.  all  growth 

FIG.  51. 


Pneumococcus  from  bouillon  culture,  resembling  streptococcus.    (Park.) 

ceases.     It  is  killed  in  a  few  minutes  by  exposure  to  a  tem- 
perature of  52°  C.    If  grown  at  42°  C.  for  twenty- four  hours, 


110  THE  PATHOGENIC  MICROCOCCL 

its  culture  becomes  very  much  attenuated,  practically  losing 
its  virulence. 

In  bouillon  it  grows  rapidly,  and  in  twenty-four  hours 
causes  a  distinct  cloudiness  of  the  medium.  At  the  end  of 
forty-eight  hours  its  growth  ceases,  and  in  four  or  five  days 
the  bouillon  becomes  clear  again,  the  bacillary  growth  being 
deposited  at  the  bottom  of  the  tube.  In  15  per  cent,  gelatin 
at  24°  C.  its  growth  is  slow.  The  gelatin  is  not  liquefied. 
On  blood-serum  at  the  temperature  of  37°  C.  it  grows  as  clear, 
almost  transparent  spots.  Its  growth  on  agar  is  very  much 
like  that  on  blood-serum.  It  does  not  grow  on  potato.  It 
causes  coagulation  of  milk. 

Immunization. — The  inoculation  of  animals  with  attenuated 
cultures  grown  at  42°  C.  for  twenty-four  hours  seems  to  protect 
the  animal  from  the  after-infection  of  virulent  cultures.  An 
infusion  made  of  the  tissues  of  immunized  animals  seems  to 
have  a  protective  influence  when  injected  simultaneously  or 
shortly  before  virulent  cultures  in  susceptible  animals. 

Pathogenesis. — Mice  and  rabbits  are  very  susceptible  to 
the  action  of  the  Micrococcus  pneumonice,  guinea-pigs  much 
less  so.  When  injected  subcutaneously  into  mice  and  rabbits, 
it  produces  a  general  septicaemia,  with  considerable  swelling 
at  the  place  of  injection  and  the  formation  of  a  fibrinous  mem- 
brane. The  spleen  is  enlarged,  and  the  bacteria  may  be  found 
in  all  the  internal  organs  and  in  the  blood,  but  no  specific 
pneumonia  is  developed.  When  intrathoracic  injections  are 
made  in  the  lung  substance,  it  produces  a  marked  lobar 
pneumonia  with  considerable  fibrinous  exudate,  and  also 
symptoms  of  general  infection.  Injected  in  the  dog  intra- 
thoracically,  it  may  produce  marked  croupous  pneumonia, 
the  animal  generally  recovering  in  two  or  three  weeks  after 
presenting  all  the  different  stages  of  the  disease. 

II.  Pneumococcus  of  Friedlaender  (Bacillus  Pneumonise 
of  Fluegge). 

The  organism  was  discovered  and  described  by  Friedlaender 
in  1883,  and  believed  by  him  to  belong  to  the  class  of  cocci, 


PNEUMONIA.  Ill 

but  recognized  afterward  as  a  bacillus.  It  is  found :  a.  in  a 
number  of  cases  of  pneumonia  in  the  fibrinous  exudate,  6.  in 
the  blood,  and  c.  in  the  sputum. 

Morphology. — Short  rods,  with  rounded  ends,  united  in 
pairs,  sometimes  in  fours,  having  a  decided  capsule  when 
taken  directly  from  the  blood  of  the  animal.  When  grown 
on  artificial  media  the  capsule  disappears.  Occasionally  the 
capsule  surrounds  each  individual  cell,  at  other  times  it  is 
around  the  cells,  united  in  pairs  or  fours.  This  capsule  may 
be  distinctly  brought  out  by  the  special  method  of  staining 
capsules  mentioned  in  the  chapter  on  staining. 

The  Bacillus  pneumonias  stains  well  with  all  anilin  dyes, 
but  does  not  stain  well  by  Gram's  method — a  diagnostic 
point  differentiating  it  from  the  Micrococcus  pneumonice. 

Biologic  Characters. — It  is  aerobic  and  facultative  anaerobic, 
non-motile,  and  has  no  flagella.  It  grows  in  all  the  media  at  a 
temperature  of  between  16°  and  20°  C.,  but  grows  best  at 
the  temperature  of  the  blood,  37°  C.  Growth  ceases  at  a 
temperature  exceeding  46°  C.  Its  growth  in  cultures  is 
exceedingly  long  lived,  so  that  after  a  year  or  longer  it  has 
grown  upon  transplantation  into  a  suitable  culture. 

Its  growth  in  bouillon  is  cloudy.  It  does  not  liquefy  gela- 
tin. Stab-cultures  in  gelatin  have  quite  a  characteristic 
appearance,  growing  in  the  form  of  a  nail.  The  head  of  the 
nail  is  at  the  point  where  the  inoculating  needle  enters  the 
gelatin,  the  path  of  the  needle  through  the  gelatin  marking 
the  body  of  the  nail.  The  head  of  the  nail  is  a  white  mass 
of  shiny  appearance ;  the  body  is  opaque  and  made  up  of 
white  spherical  colonies.  It  produces  bubbles  of  gas  in  gela- 
tin. On  gelatin  plates  colonies  appear  in  twenty-four  hours 
as  small  white  spheres  which  increase  rapidly  in  size,  and  in 
a  short  time  on  the  surface  of  the  plate  large  masses  are 
formed. 

Its  growth  on  agar  is  much  like  that  on  gelatin.  On 
blood-serum  the  growth  is  abundant,  viscid,  and  grayish  white 
in  color.  On  potato  it  grows  rapidly  and  abundantly,  and  is 
yellowish  white  in  appearance. 

Pathogenesis. — The  Bacillus  pneumonias  is  fatal  to  mice  and 


112  THE  PATHOGENIC  M1CROCOCCI. 

guinea-pigs.  Dogs  and  rabbits  are  immune.  Intrapleural 
injections  in  susceptible  animals  result  in  a  decided  pleuritic 
effusion  with  formation  of  fibrinous  membranes,  intense  con- 
gestion of  the  lungs  on  the  injected  side,  great  enlargement 
of  the  spleen,  and  general  involvement  of  the  blood  (septi- 
caemia) and  internal  organs ;  the  bacillus  being  found  every- 
where. 

EPIDEMIC  CEREBROSPINAL  MENINGITIS. 

III.  Diplococcus  Intracellularis  Meningitidis. 

This  organism  was  discovered  by  Weichselbaum,  in  1887, 
in  pus-cells  (polymorphonuclear  leucocytes)  of  the  cerebro- 
spinal  exudate  of  cases  of  epidemic  cerebrospinal  meningitis. 

Morphology. — The  micrococcus  occurs  in  bunches  or  in 
chains  of  three  or  four  elements,  the  elements  in  the  chain 
showing  marked  variation  in  size.  Stains  with  all  the  anilin 
dyes  and  is  decolorized  by  Gram's  method.  It  shows  marked 
variation  of  the  different  elements  in  their  power  of  taking 
color ;  some  elements  being  deeply  stained,  others  scarcely  at 
all.  The  organism  has  a  low  vitality ;  exposure  in  the  dry 
state  for  twenty-four  hours  to  direct  sunlight  at  the  body 
temperature,  37°  C.,  is  sufficient  to  kill  it.  At  the  room 
temperature  it  is  killed  in  seventy-two  hours  when  dried. 

To  obtain  cultures  from  man,  of  this  bacillus,  what  is  known 
as  lumbar  puncture  of  the  spine  must  be  made.  The  patient 
is  placed  on  the  left  side  very  much  in  the  same  position  as  is 
used  for  intraspinal  cocainization,  the  skin  of  the  patient  and 
hands  of  the  operator  are  thoroughly  sterilized,  and  an  ordi- 
nary antitoxin-serum  needle  is  introduced  into  the  spinal  canal, 
between  the  second  and  third  lumbar  vertebra?,  the  skin 
being  pierced  a  little  to  the  right  of  the  spinous  process.  The 
needle  is  driven  in  for  4  cm.  in  a  child,  and  7  to  8  cm.  in  an 
adult,  until  the  spinal  canal  is  reached,  when  the  spinal  fluid 
is  allowed  to  drop  into  a  clean  sterilized  test-tube.  From  5 
to  15  c.c.  of  fluid  are  generally  taken  for  examination. 
Cover-glasses  are  prepared  and  a  number  of  cultures  are  made. 
This  puncture  seems  to  be  followed  by  no  ill  effect. 


MALTA   OR  MEDITERRANEAN  FEVER.  113 

Biologic  Characters. — This  coccus  is  aerobic  and  is  a  faculta- 
tive saprophyte,  non-motile,  has  no  flagella,  and  grows  on  all 
culture-media,  but  rather  irregularly,  thriving  best  on  ordinary 
or  Loeffler's  blood-serum.  In  inoculating  cultures  from  the 
exudate  of  patients,  a  large  quantity  of  exudate  must  be  used 
and  a  number  of  tubes  inoculated,  as  otherwise  no  growth 
may  be  obtained.  It  seems  to  grow  best  when  the  exudate 
taken  comes  from  a  recent,  acute  case.  It  does  not  cloud 
bouillon,  but  causes  a  scanty  deposit  on  the  side  and  at  the 
bottom  of  the  fluid. 

On  glycerin-agar  and  blood-serum  it  grows  as  transparent, 
shiny  colonies.  It  does  not  liquefy  gelatin  nor  does  it  grow 
on  potato.  It  grows  only  at  the  temperature  of  the  body, 
37°  C.,  in  two  or  three  days.  Cultures  of  this  bacillus  live 
only  for  five  or  six  days,  so  that  it  is  necessary  to  transplant 
them  every  third  or  fourth  day. 

Pathogenesis. — It  can  not  be  inoculated  into  animals  by  the 
ordinary  methods  used,  but  intrameningeal  injections,  either 
spinal  or  under  the  cerebral  dura,  produce  a  characteristic 
meningitis  and  fibrinous  exudate,  the  bacteria  invading  at 
times  the  lungs,  but  never  being  found  in  the  blood. 

MALTA  OR  MEDITERRANEAN  FEVER. 
IV.  Micrococcus  Melitensis. 

This  organism  was  demonstrated  by  Surgeon-Major  Bruce, 
of  the  British  Army,  as  the  cause  of  what  is  known  as  Malta 
or  Mediterranean  fever. 

Morphology. — Round  or  oval  cocci  0.5  mikron  in  diameter, 
occurring  solitary  or  in  pairs,  in  cultures  occasionally  form- 
ing chains,  and  staining  by  the  usual  anilin  dyes  but  not  by 
Gram's  method. 

The  micrococcus  is  non-motile,  but  Gordon  claims  to  have 
demonstrated  the  presence  of  from  one  to  four  flagella. 

Biologic  Characters. — It  is  aerobic.  It  grows  very  scantily 
on  gelatin  at  22°  C.  only  at  the  end  of  several  weeks,  and 
does  not  liquefy  the  gelatin.  It  grows  best  in  agar,  stab 

8— M.  B. 


114  THE  PATHOGENIC  MICROCOCCL 

cultures  showing  growth  only  at  the  end  of  several  days. 
The  colonies  appear  as  pearly-white  spots  scattered  around 
the  points  of  puncture,  and  as  minute  round  white  colonies 
along  the  course  of  the  needle-track,  which  increases  in  size, 
and  after  some  weeks  a  rosette-shaped  growth  is  seen  upon 
the  surface.  Along  the  line  of  puncture  the  growth  assumes 
a  yellowish-brown  color. 

At  35°  C.  the  colonies  become  visible  only  at  the  end  of 
seven  days;  at  37°  C.  they  are  seen  in  three  or  four  days. 

It  does  not  grow  on  potato. 

Pathogenesis. — This  micrococcus  is  not  pathogenic  for  mice, 
guinea-pigs,  or  rabbits,  but  subcutaneous  injections  in  mon- 
keys have  induced  fever,  the  animal  dying  in  from  thirteen 
to  twenty-one  days.  At  the  autopsy  the  spleen  is  found 
enlarged  and  contains  the  micrococcus. 

In  man  the  micrococcus  is  found  in  the  enlarged  spleen  in 
great  numbers. 

Agglutination. — Recent  cultures  of  Micrococcus  melitensis 
are  agglutinated  by  the  blood-serum  of  patients  suffering 
from  Malta  fever,  and  occasionally  with  some  this  reaction 
is  manifested  a  year  after  recovery.  This  agglutinating  effect 
has  been  obtained  in  a  dilution  as  high  as  1  in  1000. 

QUESTIONS. 

Give  the  several  names  of  the  Micrococcus  pneumonise ;  by  whom  and  how 
was  it  discovered  ? 
Where  is  it  found  ? 
What  is  its  morphology? 
How  does  it  stain? 

How  does  it  behave  with  regard  to  oxygen  ? 
Does  it  possess  flagella  ? 
Is  it  motile  ? 

In  what  media  and  at  what  temperature  does  it  grow? 
What  is  its  thermal  death-point? 

How  does  it  grow  in  bouillon,  gelatin,  agar,  blood-serum? 
What  protects  animals  from  inoculations  with  virulent  cultures? 
What  animals  are  susceptible? 

What  are  the  effects  of  subcutaneous  and  intrathoracic  injection  of  animals  ? 
What  is  the  synonym  of  the  pneumococcus  ? 
Is  it  a  coccus  ? 
By  whom  was  it  discovered  ? 
Where  is  it  found  ? 
Give  its  morphology.      Its  staining  properties.      Give  its   principal  bio- 


PLATE  II. 


V 


Tuberculous    Sputum   Stained   by   Gabbett's    Method.     Tubercle 
Bacilli  seen  as  Red  Rods;   all  else  is  Stained  Blue.    (Abbott.) 


TUBERCULOSIS.  115 

logical  characters.  How  does  it  grow  in  bouillon,  in  gelatin,  on  agar,  on 
blood-serum,  on  potato? 

What  animals  are  susceptible? 

Describe  the  effects  of  subcutaneous  or  intrathoracic  inoculations. 

How  is  it  differentiated  from  the  preceding  germ? 

Where  is  the  Diplococcus  intracellularis  meningitidis  found? 

By  whom  was  it  discovered? 

Give  its  morphology,  its  staining  properties,  its  principal  biologic  charac- 
ters? 

How  is  lumbar  puncture  performed? 

What  animals  are  susceptible? 

How  and  where  should  the  inoculation  be  performed? 

Who  discovered  the  Micrococcus  melitensis  f 

Where  was  it  found  ''. 

State  its  morphology,  staining,  its  biologic  characters. 

What  animals  are  susceptible  ? 

In  what  dilution  does  the  blood  of  cases  of  Malta  fever  agglutinate  cult- 
ures of  this  micrococcus? 


CHAPTER    X. 

TUBERCULOSIS. 
Bacillus  Tuberculosis. 

History. — That  tuberculosis,  the  scourge  of  the  human 
race,  was  caused  by  a  microorganism,  had  long  been  sus- 
pected there  is  no  doubt,  but  it  was  not  until  Koch's  dis- 
covery of  the  bacillus  tuberculosis  in  1882  that  this  was  at 
all  proved.  (Plate  II.) 

Morphology. — The  Bacillus  tuberculosis  is  a  strict  parasite. 
It  is  aerobic  and  grows  at  the  temperature  of  the  human 
body.  It  is  a  slender  rod  from  1.5  to  3.5  mikrons  in  length, 
and  from  0.2  to  0.5  mikron  in  breadth,  occurring  singly  or  in 
pairs  united  by  their  narrow  extremities. 

It  is  found  in  all  tuberculous  growths  and  secretions,  but 
especially  in  the  sputum  of  tuberculous  patients,  where  its 
presence  is  the  best  confirmatory  evidence  of  the  existence 
of  the  disease. 

Biologic  Characters. — It  grows  with  difficulty  on  any  of 
the  artificial  media.  Koch  succeeded  in  growing  it  on  blood- 
serum.  It  does  not  grow  in  gelatin.  It  thrives  best  on  8 
per  cent,  glycerin-agar  or,  in  the  mixture  of  Roux  and 


116  TUBERCULOSIS. 

Nocard,  8  per  cent,  glycerin-bouillon.  In  this  bouillon,  kept 
at  a  temperature  of  37°  C.,  at  the  end  of  from  twelve  to 
fourteen  days  it  forms  a  small  pellicle  on  the  surface. 

In  slant  cultures  of  glycerin-agar  and  blood-serum  it  grows 
over  the  surface  of  the  medium  as  a  dried-up,  scaly-looking 
mass.  According  to  some  authorities,  it  is  a  spore-bearing 
bacterium  ;  others  fail  to  find  the  existence  of  spores  in  it. 
It  is  non-motile,  though  occasionally  slight  movements  have 
been  detected  in  it.  It  appears  to  have  no  flagella.  It  is 
usually  killed  by  exposure  to  70°  C.,  but  in  the  dried  state 
may  be  preserved  alive  for  a  considerable  time  even  at  a  tem- 
perature approaching  100°  C. 

Staining. — It  is  difficult  to  stain  by  the  usual  staining 
methods,  and  requires  the  use  of  special  staining  technic. 
Koch's  method  of  staining  it  consists  in  adding  liquor  potassse 
to  the  alkaline  anilin  dyes. 

Ehrlich's  modification  of  Koch's  method,  which  consists  in 
preparing  anilin  water  and  adding  this  to  the  solution  of  an 
anilin  dye,  is  perhaps  the  best  method  of  bringing  out  the 
tubercle  bacillus. 

The  mode  of  procedure  for  the  staining  of  bacilli  in  secre- 
tions, especially  in  sputum,  has  been  described  in  the  chapter 
on  staining,  as  the  Koch-Ehrlich  method,  or'the  Ziehl  carbol- 
fuchsin  method,  or,  better  still,  as  Gabbett's  modification  of 
Ziehl's  method. 

In  tissue  the  bacillus  is  stained  best  by  an  application  of 
either  method,  which  will  also  be  found  described  in  the  chap- 
ter on  staining. 

When  so  stained,  the  bacillus  shows  a  number  of  unstained 
places  in  the  cell-body,  somewhat  resembling  spores.  They 
have  given  rise  to  the  opinion  that  the  bacilli  are  spore-form- 
ing, but  the  fact  that  when  the  usual  method  for  staining 
spores  is  applied  these  spots  remain  unstained  seems  to  prove 
that  they  are  not  spores,  but  are  due  possibly  to  some  degen- 
eration in  the  protoplasm  of  the  bacillus. 

Nature  and  Occurrence. — As  mentioned,  the  tubercle  bacillus 
is  a  strict  parasite,  and  is  found  only  in  tuberculous  tissues 
and  in  the  secretions  from  tuberculous  patients,  especially  the 


BACILLUS  TUBERCULOSIS.  117 

sputum.  It  is  also  found  in  substances  that  have  been  con- 
taminated with  those  secretions,  and  occasionally  are  wafted 
in  the  air  in  this  manner. 

Pathogenesis. — The  tubercle  bacillus  is  pathogenic  for  man 
and  for  nearly  all  the  lower  animals,  especially  the  herbivora, 
though  the  carnivora  and  birds  are  alike  susceptible  to  it,  and 
traces  of  the  disease  have  even  been  found  in  cold-blooded 
animals.  It  may  infect  the  whole  animal  economy,  giving 
rise  to  local  manifestations  in  the  shape  of  nodules  whicli 
contain  the  bacillus. 

The  usual  mode  of  infection  of  animals  is  through  the  re- 
spiratory tract,  but  sometimes  through  the  gastro-intestinal 
tract.  Infection  may  occasionally  be  produced  by  the  intro- 
duction of  the  bacilli  through  abrasions  of  the  skin,  as  in  the 
case  of  dissectors  or  pathologists,  when  it  gives  rise  to  local- 
ized tuberculous  nodules  on  the  hands,  which  at  any  time 
may  become  the  source  of  infection  of  the  general  organism. 

The  usual  mode  of  inoculation  of  animals  is  either  by  intra- 
peritoneal  inoculation,  when  it  gives  rise  to  a  general  tubercu- 
losis involving  especially  the  glands  of  the  abdomen  and  the 
lungs,  or  by  subcutaneous  inoculation,  when  a  small  quantity 
of  the  culture  or  a  small  bit  of  the  suspected  substance  is 
used. 

The  usual  contaminating  substance  for  man  is  the  secretion 
of  tuberculous  patients,  which  may  be  deposited  on  utensils 
used  by  others,  or  which  through  carelessness  may  have  dried 
in  the  room,  thus  contaminating  the  dust  of  the  apartment, 
which,  wafted  through  the  air,  is  brought  into  contact  with  the 
mucous  membrane  of  the  respiratory  organs  of  susceptible 
individuals.  In  this  way  the  air  of  hospital  wards  of  con- 
sumptives and  the  various  articles  of  furniture  in  rooms 
inhabited  by  consumptives  have  been  proved  to  be  infec- 
tious. 

The  drinking  of  contaminated  milk  and  the  eating  of  meat 
from  tuberculous  animals  are  believed  in  some  instances  to 
have  spread  the  disease.  This,  however,  is  not  thoroughly 
proved,  and  recently  the  eminent  Koch  has  asserted  that  this 
mode  of  contamination  is  exceedingly  rare,  and  is  an  equation 


118  TUBERCULOSIS. 

which  in  the  treatment  and  the  prevention  of  tuberculosis 
may  be  altogether  neglected. 

It  has  been  assumed  that  human,  avian,  and  bovine  tuber- 
culosis are  identical.  In  a  remarkable  paper  on  tuberculosis 
by  Koch,  read  before  the  Tuberculosis  Congress  in  1901,  at 
Berlin,  he  denies  this  identity,  and  shows  by  a  number  of 
experiments  that  cattle  can  not  be  inoculated  with  the  secre- 
tion of  tuberculous  patients,  and  that  man  is  not  affected  by 
eating  meat  from  contaminated  oxen. 

As  regards  the  transmission  of  tuberculosis,  the  part  played 
by  heredity  is  almost  nil.  It  has  failed  of  demonstration 
that  foetuses  or  young  children  from  intensely  tuberculous 
mothers  have  in  their  secretions  or  tissues  the  tubercle  bacil- 
lus ;  and  reasoning  by  analogy,  as  in  Bang's  method,  the  sepa- 
ration of  newborn  calves  from  their  tuberculous  mothers  has 
completely  succeeded  in  eliminating  tuberculous  diseases 
from  these  calves,  it  must  be  assumed  that  like  precautions 
would  produce  identical  results  in  man. 

The  tubercle  bacilli  secrete  a  poisonous  material,  which  is 
chiefly  contained  in  the  bacterial  cells  themselves,  and  is 
known  by  the  name  of  tuberculin.  This  tuberculin  is  believed 
to  be  a  preventative  against  tubercular  diseases  ;  and  in  1890 
Koch  proclaimed  that  by  means  of  injections  of  this  substance 
he. had  succeeded  in  curing  tuberculosis.  This  promise  has  not 
been  fully  realized,  but  Koch's  discovery  has  given  us  valu- 
able information,  and  has  demonstrated  that  by  injection  of 
this  tuberculin  healthy  animals  may  be  recognized  and  so 
separated  from  tuberculous  ones  long  before  the  disease  could 
be  diagnosed  in  the  latter  by  physical  signs  ;  for  the  former 
are  not  affected  by  small  doses  of  tuberculin,  whereas  animals 
that  have  the  least  tuberculous  taint  will  show  decided  reac- 
tion when  injected  with  tuberculin.  This  procedure  is  used 
extensively  in  all  civilized  countries  nowadays  for  the  diag- 
nosis of  tuberculosis  in  cattle  and  other  animals. 

The  original  tuberculin  of  Koch  is  prepared  from  an  extract 
of  glycerin-bouillon  of  virulent  bacteria,  in  which  the  bacteria 
themselves  are  quickly  killed  by  exposure  to  a  higher  tem- 
perature, and  filtered  away  by  a  Chamberlain  filter.  0.025 


BACILLUS  TUBERCULOSIS.  119 

c.c.  of  such  an  extract  will  in  tuberculous  animals  develop 
marked  reactionary  symptoms,  whereas  when  used  in  healthy 
animals  it  gives  rise  to  no  reaction. 

This  tuberculin  has  a  beneficial  action  in  man,  especially  an 
action  on  local  tuberculous  diseases,  such  as  lupus,  tuberculous 
joints,  etc.  It  is  dangerous,  however,  when  used  therapeu- 
tical ly,  because  it  shows  a  tendency  to  stimulate  the  develop- 
ment of  dormant  tuberculosis. 

Recently  different  forms  of  tuberculin  have  been  prepared 
by  Koch,  known  as  tuberculin  A,  O,  and  R. 

Tuberculin  A. — This  is  prepared  by  extracting  the  bacilli 
with  decinormal  salt  solution,  and  acts  very  much  like  ordi- 
nary tuberculin,  being  even  more  severe  in  effect. 

Tuberculin  0. — This  is  prepared  by  pounding  the  dried 
tubercle  bacilli  and  extracting  with  distilled  water,  the 
emulsion  being  then  passed  through  the  centrifuge.  The 
residue  after  centrifugation  is  dried  and  again  pounded  and 
extracted  with  water,  and  these  processes  repeated  until  no 
solid  residue  is  left.  The  whitish  liquids  from  all  the'se 
operations  are  mixed,  and  the  result  is  tuberculin  R. 

Tuberculin  O  is  identical  in  effect  to  tuberculin  A  and 
has  an  immunizing  effect.  Tuberculin  R  gives  rise  to  little 
reaction,  but  has  a  decided  immunizing  effect.  The  fluid 
in  tuberculin  R  is  made  so  that  1  c.c.  corresponds  to 
10  milligrams  of  solid  matter,  and  must  be  diluted  with 
sterile  salt  solution  to  bring  it  to  the  required  strength.  In 
applying  the  same  therapeutically  the  dose  of  tuberculin  R 
for  an  adult  is  -^^  to  1  milligram.  It  must  be  used  hypo- 
dermatically,  and  should  be  administered  every  other  day. 
The  dose  should  not  give  rise  to  a  temperature  exceeding 
1  degree  C. 

This  produces  very  satisfactory  results  in  the  treatment 
of  lupus,  but  so  far  in  tuberculous  diseases  of  the  lung  its 
effects  have  not  come  up  to  expectation. 

Recently,  the  tubercle  bacillus,  on  account  of  its  peculiai 
growth  in  some  cases  in  which  it  seems  to  present  projecting  proc- 
esses or  branches,  has  been  thought  by  some  to  belong  to  the 


120  LEPROSY  AND  SYPHILIS. 

higher  bacteria,  being  probably  a  streptothrix,  closely  related  to 
the  actinomyces. 

QUESTIONS. 

When  and  by  whom  was  the  Bacillus  tuberculosis  discovered  ?  How  does 
the  bacillus  behave  in  the  presence  of  oxygen  ? 

What  is  the  size  of  the  tubercle  bacillus?  In  what  tissues  and  secretions 
of  tuberculous  animals  is  it  usually  found  ?  How  is  it  best  artificially  grown  ? 
What  temperature  is  most  favorable  fur  its  growth?  How  high  a  temperature 
does  it  resist? 

Give  two  methods  of  staining  the  tubercle  bacillus  in  cultures  or  in  the  se- 
cretions of  animals.  Give  the  mode  of  staining  the  bacteria  in  tissue?  What 
has  given  rise  to  the  idea  of  spores  in  the  bacillus? 

What  animals  besides  man  are  the  most  susceptible  to  tuberculous  diseases? 
What  two  forms  of  infection  follow  inoculation  of  this  bacillus?  What  is  the 
usual  mode  of  infection  in  man  ? 

Mention  some  cases  of  localized  tuberculosis  in  man.  How  are  animals  in- 
oculated to  produce  the  disease  ?  What  are  the  usual  infecting  agents  in  man  ? 

What  part  does  tuberculous  milk  or  tuberculous  meat  play  in  the  dissem- 
ination of  tuberculosis? 

What  was  the  subject  of  Koch's  paper  at  the  Congress  of  Tuberculosis, 
in  1901? 

What  part  does  heredity  play  in  the  transmission  of  tuberculosis? 

What  is  tuberculin  ? 

What  diagnostic  purpose  does  tuberculin  serve  ? 
•  How  is  tuberculin  prepared  ? 

What  is  meant  by  Tuberculin  A,  O,  and  E  ? 

Why  has  the  tubercle  bacillus  been  thought  to  be  a  streptothrix? 


CHAPTER  XI. 

LEPROSY  AND  SYPHILIS. 

LEPROSY. 
Bacillus  Leprae. 

History. — The  specific  cause  of  leprosy  is  a  bacillus  known 
as  the  Bacillus  leprce,  discovered  by  Hansen,  and  confirmed  by 
Neisser,  in  1879. 

The  bacillus  is  found  a.  in  the  tissues  of  leprous  patients, 
and  b.  in  the  secretions,  with  the  exception  of  the  urine.  It 
has  never  been  found  in  the  blood. 

Morphology. — The  bacilli  are  small  straight  rods  with 
pointed  ends,  sometimes  curved,  measuring  from  5  to  6  mi- 


LEPROSY.  121 

krons  in  length,  non-motile,  resembling  very  much  the  tubercle 
bacillus,  but  are  more  uniform  in  length  and  not  so  frequently 
bent.  When  stained,  their  protoplasm  shows  unstained  spaces 
similar  to  those  of  the  tubercle  bacillus,  which  are  regarded 
by  some  as  spores. 

Biology. — Bordoni-Uffreduzzi  claims  to  have  cultivated  the 
bacillus  through  a  number  of  generations  in  glycerinized  gela- 
tin. Byron  (Researches  Loomis  Laboratory,  1892)  made  a 
pure  culture  of  the  bacillus  on  agar. 

From  the  secretions  and  scrapings  obtained  from  an  ulcer 
of  the  nares  in  a  leper  the  author  found  upon  examination  a 
great  many  bacilli  lying  in  cells,  some  cells  containing  as 
many  as  3  or  4  bunches,  and  was  able  to  procure  a  pure 
culture  on  Loeffler's  blood-serum  and  glycerin-agar. 

The  growth  upon  the  serum  very  much  resembled  a  twisted 
band  of  yellowish-gray  color,  and  developed  very  rapidly  at 
37°  C.  Cultures  in  bouillon  and  potato  did  not  develop. 

The  Bacillus  leprce  stains  very  readily  with  the  anilin  dyes, 
and  also  by  Gram's  method.  It  very  greatly  resembles  the 
tubercle  bacillus  in  retaining  its  color  when  subsequently 
treated  with  strong  solutions  of  mineral  acids. 

An  interesting  point  about  the  staining  of  the  Bacillus 
leprce  which  will  permit  differentiation  from  the  Bacillus  tuber- 
culosis is  that  the  lepra  bacillus  is  rapidly  stained  by  the 
Gram  method,  while  the  tubercle  bacillus  stains  with  great 
difficulty  by  it,  and  must  remain  at  least  twenty-four  hours 
in  the  color  dish  before  taking  the  stain. 

Baumgarten's  differentiation  between  these  two  bacteria  is 
to  subject  cover-glass  preparations  which  have  been  smeared 
with  scrapings  from  leprous  nodules  or  ulcers  for  five  minutes 
in  the  Ehrlich  solution,  and  afterward  to  decolorize  with  solu- 
tion of  nitric  acid  in  alcohol,  1  part  of  acid  to  10  parts  of 
alcohol.  The  bacillus  of  Hansen  will  be  stained,  while  the 
tubercle  bacillus  will  not. 

A  number  of  investigators  have  by  inoculation  with  fresh 
extirpated  leprous  tissue  succeeded  in  reproduei^gthe  disease 
in  the  lower  animals.  Tedeschi  inoculated  a  monkey  under 
the  dura  mater,  and  death  resulted  in  six  days.  Many  lepra 


122  LEPROSY  AND  SYPHILIS. 

bacilli  were  found  in  the  spleen  and  spinal  cord  at  the 
autopsy. 

Nature  of  Leprosy. — Besnier,  with  many  others,  contends 
that  leprosy  is  a  bacterial  disease  exclusively  limited  to  man, 
and  that  the  microorganisms  will  reproduce  themselves  in 
man  alone,  and  not  in  animals. 

Dyer,  from  observation  of  leprosy  in  fifty  cases  in  Louis- 
iana, concludes  positively  that  the  direct  cause  of  the  disease 
is  the  lepra  bacillus.  The  indirect  cause  is  contagion.  The 
disease  therefore  is  not  hereditary. 

A  very  useful  method  of  diagnosis  for  physicians  who  wish 
to  make  a  speedy  and  positive  proof  of  leprosy,  and  have  no 
microtome  or  laboratory  facilities,  is  to  remove  a  bit  of  skin 
or  scraping  near  a  tubercle  or  nodule  and  place  the  same  in  a 
mortar  with  some  saline  solution  and  triturate  until  a  homo- 
geneous solution  results,  adding  from  time  to  time  enough 
saline  solution  to  prevent  drying.  A  small  quantity  of  this 
emulsion  is  transferred  to  a  clean  cover-glass,  air-dried,  and 
fixed  over  a  flame,  stained  with  the  Ziehl  carbol-fuchsin  for 
five  minutes,  then  washed  in  water,  counterstained,  and  de- 
colorized with  Gabbett's  solution  of  methylene-blue  and  sul- 
phuric acid  for  two  minutes,  washed  again  in  water,  dried, 
and  mounted  in  Canada  balsam.  The  bacilli  will  appear  red, 
while  the  rest  of  the  tissue  will  be  stained  blue. 

SYPHILIS. 

Bacillus  of  Syphilis. 

History. — In  1884-1885  Lustgarten  described  a  bacillus 
which  he  had  discovered  in  the  primary  sore  and  secondary 
manifestations  of  syphilis.  Rarely  could  this  bacillus  be 
found  in  the  tertiary  stages  of  the  disease. 

In  size  and  shape  the  bacillus  very  closely  resembles  that 
of  tuberculosis,  but  differs  from  it  especially  in  its  cultural 
peculiarities  and  also  in  its  staining  properties  with  the  anilin 
dyes.  For  instance,  the  bacillus  could  not  be  cultivated  on 
any  of  the  artificial  media,  not  even  on  those  on  which  the 
Bacillus  tuberculosis  could  be  made  to  grow ;  and  in  staining 


QUESTIONS.  123 

the  Bacillus  syphilidis  it  showed  considerable  difficulty  in  tak- 
ing up  the  anilin  colors,  yet  when  stained  according  to  Ehr- 
lich's  or  ZiehPs  method  it  very  quickly  parted  with  its  colors 
when  washed  in  mineral  acids,  especially  sulphuric  acid,  con- 
trary to  what  happens  in  the  case  of  the  Bacillus  tuberculosis. 
When  decolorized,  however,  by  means  of  alcohol  the  Bacillus 
syphilidis  retained  the  dye  for  a  considerable  time. 

For  the  staining  of  sections  the  following  method  is  recom- 
mended :  Place  the  section  in  a  cold  solution  of  anilin- water 
gentian-violet  for  from  twelve  to  twenty-four  hours  at  the 
room  temperature,  or  for  two  hours  at  a  temperature  of 
40°  C.  Wash  a  few  minutes  in  absolute  alcohol,  then  put 
the  section  for  some  seconds  into  1.5  per  cent,  solution  of  per- 
manganate of  potassium,  pass  rapidly  (for  one  or  two  seconds) 
into  sulphuric  acid  solution,  wash  thoroughly  in  water,  and 
mount  on  xylol  balsam.  When  stained  by  this  method  the 
Bacillus  syphilidis  shows  considerable  resemblance  to  the 
Bacillus  tuberculosis,  being  of  similar  size  and  showing  similar 
refractive  spots  in  the  body  of  the  cell. 

As  mentioned  above,  this  bacillus  has  not  been  successfully 
cultivated  artificially,  and  inoculations  of  animals  have  also 
been  barren  of  results. 

Streptococcus  of  Syphilis. 

Vanniessen,  by  collecting  blood  of  syphilitic  subjects,  and 
allowing  same  to  coagulate  in  sterilized  tubes,  has  been  able 
from  the  serum  of  this  blood  to  cultivate  a  streptococcus  which 
he  believes  to  be  the  etiological  factor  in  syphilis.  His 
experiments,  however,  have  failed  of  confirmation  by  others. 

QUESTIONS. 

When  and  by  whom  was  the  Bacillus  leprse  found  ?    Where  is  it  found  ? 

Describe  the  Bacillus  leprse.  Does  it  contain  spores?  How  is  it  grown 
artificially  ?  How  does  it  stain  ? 

How  do  you  differentiate  Bacillus  leprse  from  the  Bacillus  tuberculosis  by 
staining?  What  animals  are  susceptible  to  the  infection? 

Give  a  ready  method  for  the  diagnosis  of  a  leprous  ulcer  or  nodule,  and 
give  a  diagnosis  of  leprosy  in  a  suspected  case. 

Describe  the  Bacillus  syphilidis  of  Lustgarten  ?  Where  is  it  found  ?  How 
does  it  stain?  How  decolorized?  How  does  it  stain  in  tissue?  How  does  it 
grow  in  artificial  culture-media  ? 

Differentiate  between  Bacillus  syphilidis  and  Bacillus  tuberculosis. 


124  LEPROSY  AND  SYPHILIS. 

CHAPTER   XII. 

GLANDERS  (FARCY). 

Bacillus  Mallei. 

GLANDERS  is  a  disease  of  the  horse  and  ass  tribe,  charac- 
terized by  the  formation  of  nodules  in  the  mucous  membrane 
of  the  mouth  and  respiratory  passages.  These  nodules,  very 
prone  to  ulcerate,  give  rise  to  profuse  suppuration,  and  very 
soon  afterward  the  lymphatic  glands  of  the  neck  begin  to 
enlarge.  These  glands  soften  early  and  discharge  a  very 
virulent  pus.  Secondarily  the  lungs  become  infected,  the 
infectious  material  forming  small  nodules  very  much  resem- 
bling tubercles  in  appearance. 

History. — In  1882,  Loeffler  discovered  in  the  discharges  and 
tissues  of  animals  affected  with  this  disease  a  specific  micro- 
organism which  he  called  Bacillus  mallei. 

Morphology. — Glanders  bacillus  is  a  bacillus  with  rounded 
or  pointed  ends,  occurring  generally  singly,  occasionally  in 
pairs,  seldom  or  never  forming  threads.  The  bacillus  is  non- 
motile,  and  therefore  possesses  no  flagella. 

Spores. — Some  observers  claim  to  have  discovered  spores  in 
the  glanders  bacillus,  but,  reasoning  by  analogy,  those  shiny 
particles  described  as  spores  are  really  not  spores ;  they  are 
the  same  as  the  shiny  particles  discovered  in  stained  prepara- 
tions of  the  Bacillus  tuberculosis,  and  they  cannot  be  stained 
by  the  usual  methods  of  spore-staining,  nor  do  the  bacteria 
containing  same  resist  conditions  which  are  usually  resisted 
by  other  spore-bearing  bacteria.  The  observation  of  Loeffler, 
however,  that  this  microorganism  is  able  to  grow  after  being 
kept  in  the  dry  state  for  a  long  time,  makes  it  appear  as  if 
some  form  of  permanent  spore  existed. 

Biology. — The  Bacillus  mallei  grows  readily  on  all  ordinary 
media  at  a  temperature  between  25°  and  38°  C.  Its  growth 
is  very  slow,  arid  on  this  account  its  isolation  and  cultivation 


GLANDERS. 


125 


by  the  usual  plate-methods  are  rather  difficult.  Upon  nutrient 
agar  it  appears  as  a  moist  opaque  layer.  On  gelatin  its  growth 
is  much  less  voluminous  than  on  agar.  It  does  not  liquefy 
the  gelatin.  In  blood-serum  the  growth  is  opaque,  moist,  of 
a  bright-yellow  color  ;  the  serum  is  not  liquefied.  On  potato 
at  37°  C.  its  growth  is  rapid,  moist,  and  of  an  amber-yellow 
color,  which  becomes  darker  with  age  and  finally  becomes  of 
a  reddish-brown.  It  causes  clouding  of  bouillon,  with  a 
tenacious,  ropy  sediment.  In  litmus  milk  it  produces  acidity 

FIG.  52. 


Bacillus  of  glanders  (Bacillus  mallei),  from  culture.    (Abbott.) 

in  four  or  five  days,  as  seen  by  the  change  of  color  from  blue 
to  red.     It  also  causes  coagulation  of  the  milk. 

Bacillus  mallei  is  very  susceptible  to  the  effect  of  high  tem- 
perature. At  40°  C.  it  will  grow  for  twenty  or  more  days. 
It  will  not  grow  at  43°  C.,  and  if  exposed  to  that  temperature 
for  forty-eight  hours  it  is  destroyed.  It  is  killed  by  a  tem- 
perature of  50°  C.  in  five  hours,  and  does  not  survive  more 
than  five  minutes  at  a  temperature  of  55°  C.  It  is  aerobic 
and  facultative  anaerobic. 


126  LEPROSY  AND  SYPHILIS. 

It  stains  readily  with  all  the  anilin  dyes,  but  presents  in 
its  body  conspicuous  irregularity  of  staining,  showing  places 
stained  very  deeply  and  others  that  have  scarcely  any  dye 
at  all. 

It  is  difficult  to  stain  in  tissues  from  the  fact  that,  though 
readily  stained,  the  bacillus  parts  very  quickly  with  its  color- 
ing-matter in  the  presence  of  a  decolorizing  agent,  and  even 
in  the  alcohol  used  to  dehydrate  the  tissue. 

A  number  of  methods  for  staining  sections  of  tissue  for  the 
bacillus  of  glanders  have  been  suggested.  The  following  is 
the  best : 

Transfer  the  sections  from  alcohol  to  distilled  water,  put 
the  sections  upon  a  slide  and  absorb  the  water  with  blotting- 
paper,  stain  for  a  half-hour  with  a  few  drops  of  a  10  per  cent, 
solution  of  carbol-fuchsin  in  water,  remove  the  superfluous 
stain  with  blotting-paper,  wash  the  sections  three  times  in  a 
0.3  per  cent,  acetic  acid  solution,  not  allowing  the  acid  to  act 
more  than  ten  seconds  each  time,  and  remove  the  acid  by  care- 
fully washing  with  distilled  water.  Absorb  all  water  with 
blotting-paper,  and  heat  moderately  over  the  flame  so  as  to 
drive  off  the  remaining  water.  Clear  in  xylol  and  mount  in 
xylol  balsam.  In  properly  stained  tissues  the  bacilli  will  be 
found  more  numerous  in  the  centre  of  the  nodule,  becoming 
fewer  as  the  periphery  is  approached. 

The  animals  susceptible  to  infection  by  glanders,  besides 
horses  and  asses,  are  guinea-pigs,  cats,  and  field-mice.  The 
rabbit  is  very  little  so  ;  dogs  and  sheep  still  less  so.  Man  is 
susceptible,  and  not  seldom  the  infection  terminates  fatally. 
House-mice,  rats,  cattle,  and  hogs  are  insusceptible. 

For  inoculation  experiments  the  guinea-pig  is  made  use  of. 
The  experiment  is  generally  performed  by  subcutaneous  inocu- 
lation of  the  culture  or  a  small  piece  of  the  nodule  from 
the  diseased  animal.  The  most  prominent  symptom  in  the 
animal  is  the  enlargement  of  the  spleen,  with  formation  of 
nodules  in  that  organ  and  in  the  liver.  From  these  nodules 
the  glanders  bacillus  may  be  obtained  in  pure  culture.  The 
animals  live  from  six  to  eight  weeks.  The  specific  character 
of  the  inflammation  of  the  mucous  membrane  of  the  nostrils 


QUESTIONS.  127 

is  almost  always  present.  The  joints  become  swollen  and 
the  testicles  enormously  distended  ;  the  internal  organs — 
lungs,  kidney,  spleen,  and  liver — are  the  seats  of  the  nodular 
deposits,  from  which  bacilli  may  be  obtained  in  pure  cultures. 

Diagnosis. — The  method  of  Strauss  for  the  recognition  of 
the  disease  is  of  great  importance  clinically.  With  it  in  a 
short  time  a  diagnosis  may  be  arrived  at,  while  by  the  ordi- 
nary methods  of  inoculation  it  would  take  weeks  to  come  to 
a  certain  conclusion.  Its  details  are  these  :  Into  the  perito- 
neal cavity  of  a  male  guinea-pig  a  bit  of  the  suspected 
tissue  is  introduced.  If  the  case  be  one  of  glanders,  in  about 
thirty  hours  the  testicles  begin  to  swell  and  the  skin  covering 
them  becomes  red  and  shining,  and  there  is  evidence  of  ab- 
scess-formation. T/ie  tumefaction  of  the  testicle  is  a  true 
diagnostic  sign. 

Mallein,  the  toxic  principle  secreted  by  the  bacillus,  has  been 
isolated  from  old  glycerin-bouillon  cultures  of  the  Bacillus  mallei. 
For  this  purpose  the  cultures  are  steamed  in  a  sterilizer  for 
several  hours  and  then  filtrated  through  a  Chamberlain  por- 
celain filter  and  evaporated  to  one-tenth  of  their  volume. 

This  mallein  is  used  as  a  diagnostic  test  for  glanders  in 
animals,  very  much  as  tuberculin  is  for  tuberculosis.  It 
produces  when  injected  in  very  small  quantity  a  rise  of  a 
degree  and  a  half  C.  if  the  animal  be  at  all  infected  with  the 
disease,  but  in  healthy  animals  injection  is  followed  by  no 
febrile  reaction. 

Some  observers  have  asserted  that  the  injection  of  this 
mallein  into  susceptible  animals  will  protect  them  from  the 
disease ;  other  observers  assert  that  the  blood-serum  of  nat- 
urally immune  animals  is  curative  when  injected  into  infected 
animals.  But  these  points  are  not  fully  determined. 

QUESTIONS. 

What  are  the  synonyms  for  the  hacillus  of  glanders? 

What  are  the  symptoms  of  glanders  in  the  horse  ? 

By  whom  and  when  was  this  bacillus  discovered? 

Describe  the  bacillus. 

Does  it  contain  spores? 

Give  reasons  for  and  against  its  spore-formation. 


128  ANTHRAX. 

What  are  its  cultural  peculiarities,  if  any,  on  agar,  on  gelatin,  on  potato, 
on  blood-seruin,  in  bouillon,  in  litmus  milk? 

Give  a  method  of  staining  the  glanders  bacillus  in  tissue. 

Give  the  method  of  inoculation  of  a  guinea-pig,  and  the  prominent 
symptoms. 

How  long  does  the  animal  live  ? 

Give  Strauss'  method  of  inoculation  for  diagnosis. 

What  is  mallein  ?  How  is  it  obtained  ?  What  are  its  uses  ?  Does  it  pro- 
tect from  glanders  ? 


CHAPTER  XIII. 

ANTHRAX. 
Bacillus  Anthracis. 

History. — The  Bacillus  anthracis,  discovered  and  described 
by  Davaine,  in  1868,  is  the  first  bacillus  that  was  demon- 
strated to  be  pathogenic  to  man  and  animals. 

It  is  found  in  the  blood  and  tissues  of  animals  which  have 
died  of  this  disease,  which  is  known  as  splenic  fever,  and 
charbon. 

It  produces  in  these  animals  a  genuine  septicaemia,  the 
capillaries  all  over  the  body  teeming  with  the  microorganisms. 

No  bacteria  have  more  than  the  Bacillus  anthracis  helped 
to  establish  the  three  postulates  of  Koch  used  in  testing  the 
pathogenicity  of  bacteria.  These  postulates  are  as  follows  : 

I.  For  a  microorganism  to  be  considered  the  cause  of  a  dis- 
ease, it  must  at  all  times  be  found  in  the  organs,  blood,  or 
secretions  of  an  animal  dead  or  affected  with  the  disease. 

II.  It  must  be  possible  to  isolate  this  organism  and  obtain 
it  in  pure  cultures  from  the  same  sources.     It  may  also  be 
grown  for  several  generations  in  artificial  culture -media. 

III.  Inoculation  of  these  pure  cultures  into  susceptible  animals 
must  give  rise  to  the  same  symptoms  and  changes  found  in 
the  animal  originally  affected,  and  the  same  bacteria  must  be 
found  in  their  blood,  tissues,  or  secretions. 

Morphology. — The  anthrax  bacillus  is  a  rod  bacterium 
measuring  from  2  to  3  mikrons  when  found  in  the  blood  and 


BACILLUS  AXTHHAC1S.  129 

tissues  of  animals ;  from  20  to  25  mikrons  when  obtained 
from  cultures;  and  of  a  uniform  thickness  of  1.25  mikrons. 
The  ends  of  the  rod  seem  a  little  thicker  than  the  rest  of  the 
body,  and  under  a  low  power  look  square,  but  with  a  higher 
power  they  are  seen  to  be  concave  (Fig.  53). 

FIG.  53. 


Bacillus  anthracis,  highly  magnified  to  show  swellings  and  concavities  at  extremities 
of  the  single  cells.    (Abbott.) 

It  is  found  singly  or  in  pairs  in  the  blood  and  tissues  of 
diseased  animals,  but  when  cultivated  in  bouillon  or  in  the 
hanging  drop  it  forms  long  threads  which  may  or  may  not 
contain  spores. 

It  is  stained  by  all  the  alkaline   anilin   dyes,  the   spores 

FIG.  54. 


Threads  of  Bacillus  anthracis  containing  spores.    X  about  1200.    (Abbott.) 

remaining  uncolored  ;  but  the  latter  are  easily  stained  by  any 
of  the  special  methods  for  staining  spores  described  in  the 
chapter  on  staining. 

Biologic  Characters. — The  Bacillus  anthracis   is  anaerobic, 
but  can  grow  without  the  presence  of  oxygen.     When  grown 
9— M.  B. 


130  ANTHRAX. 

with  free  access  of  oxygen  in  artificial  culture-media  it  forms 
long  filaments  or  threads,  which  are  formed  by  the  union  of  a 
number  of  bacilli.  In  the  presence  of  free  oxygen  elliptical 
bright  spots,  one  to  each  segment  of  the  thread,  are  observed; 
these  are  the  spores. 

This  bacillus  grows  at  all  temperatures  between  12°  and 
45°  C.,  but  it  does  not  form  spores  at  a  temperature  below 
18°  or  above  42°  C.  Its  maximum  of  growth  is  at  37.5°  C. 
(Fig.  54). 

In  the  blood  and  tissues  of  animals  it  does  not  sporulate. 
The  bacterium  is  non-motile  and  has  no  flagella. 

In  bouillon  it  grows  very  rapidly,  forming  twisted  thread- 
like masses,  resembling  cotton,  in  the  mass  of  the  bouillon 

FIG.  55. 


Colony  of  Bacillus  anthracis  on  agar-agar.    (Abbott.) 

and  at  the  bottom  of  the  tube,  but  it  does  not  cloud  the 
medium. 

On  agar  its  growth  is  quite  characteristic,  forming  colonies 
which  look  like  irregularly  twisted  knots  of  thread  resem- 
bling cotton-wool ;  this  peculiar  growth  has  been  given  the 
name  of  the  head  of  Medusa  (Fig.  55). 

On  gelatin  its  growth  is  very  like  that  on  agar,  but  it 
liquefies  the  medium.  On  potato  it  grows  rapidly  as  a  dull 
white,  thread-like  mass. 

Resistance  to  Thermal  Changes. — It  does  not  grow  at  a 
temperature  below  12°  C.  or  above  45°  C.  It  may,  however, 
when  containing  spores,  be  kept  for  almost  an  indefinite  period 
even  when  dried  and  exposed  to  a  high  temperature,  and  be 


BACILLUS  ANTHRAC1S.  131 

subsequently  grown  when  brought  in  a  suitable  medium. 
The  spores  resist  a  freezing  temperature,  and  even  the  tem- 
perature of  liquid  air,  for  almost  an  indefinite  time.  They 
are  killed  by  dry  heat  at  a  temperature  of  140°  C.  only  after 
three  hours'  exposure,  and  at  150°  C.  only  after  one  hour's 
exposure.  By  moist  heat  at  the  temperature  of  100°  C.  they 
are  killed  in  from  three  to  four  minutes.  They  resist  the 
action  of  5  per  cent,  carbolic  acid  for  five  minutes. 
•  Its  non-sporing  forms  are  killed  by  a  temperature  of  54°  C. 

Pathogenesis. — Cattle,  sheep,  horses,  mice,  guinea-pigs,  and 
rabbits  are  all  susceptible  to  the  action  of  the  bacilli.  Am- 
phibia, dogs,  white  rats,  and  birds  are  not  susceptible.  Sus- 
ceptible animals  may  be  infected  in  one  of  four  ways  :  through 
the  abrasions  of  the  skin  and  mucous  surfaces,  through  the 
respiratory  tract,  through  the  alimentary  tract,  or  by  subcu- 
taneous inoculation,  as  generally  practised  in  the  laboratory. 

When  the  bacillus  is  inoculated  subcutaneously  into  animals, 
the  animal  shows  little  or  no  inflammation  at  the  point  of 
inoculation,  but  marked  oedema  of  the  subcutaneous  tissue  at 
a  distance  from  the  inoculating  point,  with  small  points  of 
blood  extravasation  in  this  tissue.  To  the  naked  eye  there 
is  very  little  change  in  the  internal  organs  except  in  the  spleen, 
which  is  enlarged,  darker,  and  soft.  Bacilli  may  be  found 
everywhere  in  the  capillaries,  in  organs  and  blood,  but  espe- 
cially in  the  vessels  of  the  lungs,  the  liver,  and  in  the  glom- 
eruli  of  the  kidneys.  Death  takes  place  in  from  one  to  three 
days  according  to  the  size  of  the  animal  and  the  dose  given. 

The  most  susceptible  animal  is  the  mouse,  next  comes  the 
guinea-pig,  and  then  the  rabbit,  and  so  uniformly  is  the 
resistance  of  these  animals  shown  to  the  action  of  inocula- 
tions with  anthrax  that  the  virulence  of  attenuated  cultures 
used  for  protective  inoculations  are  tested  on  those  animals. 

Immunization. — Pasteur  has  demonstrated  that  attenuated 
cultures  of  the  Bacillus  antJtracis  when  injected  into  susceptible 
animals  are  capable  of  protecting  the  same  against  the  action 
of  the  virulent  bacillus,  subsequently  inoculated,  and  against 
an  attack  of  the  disease  itself.  His  inoculation  or  vaccination 
consists  in  using  cultures  that  have  been  attenuated  by  means 


132  ANTHRAX. 

of  heat.  For  that  purpose  the  bacteria  are  cultivated  in 
large  Erlenmeyer  flasks  at  a  temperature  of  between  42° 
and  43°  C.,  for  a  period  of  time  varying  from  ten  to  thirty 
days,  when  they  do  not  form  spores.  The  pathogenic  power 
of  these  cultures  is  tested  every  few  days  on  guinea-pigs 
and  rabbits,  and  when  a  small  dose  of  the  culture  will  kill 
a  mouse  and  a  guinea-pig,  but  fails  to  kill  a  rat,  it  is  called 
vaccine  No.  2.  In  a  few  days  more  this  same  culture  will 
fail  to  kill  a  guinea-pig,  but  will  still  kill  a  mouse ;  it  is 
then  vaccine  No.  1. 

In  veterinary  practice  large  animals,  as  sheep,  cattle,  and 
horses,  are  inoculated  with  aseptic  precautions  with  3  c.c.  of 
vaccine  No.  1.  Then  they  show  little  or  no  reaction.  In  ten 
days  or  two  weeks  more  they  are  inoculated  with  vaccine 
No.  2,  when  they  again  show  some  little  reaction  ;  and  a  few 
days  after  this  second  vaccination  they  are  able  to  withstand 
an  inoculation  of  virulent  cultures  of  the  bacilli.  This  mode 
of  vaccination  has  been  of  inestimable  value  by  making  it 
possible  to  stop  the  ravages  of  epidemics  of  anthrax.  It  is 
practised  extensively  in  countries  like  France,  Germany,  and 
Russia,  where  the  disease  is  very  prevalent  among  sheep  and 
cattle.  In  the  Southern  States  the  author  has  had  occasion 
to  use  it  extensively  during  the  last  few  years,  with  decided 
benefit. 

The  manner  of  infection  among  animals  with  the  bacilli  has 
not  been  fully  demonstrated.  It  seems  to  occur  in  the  ma- 
jority of  cases  from  the  soil,  possibly  from  the  fact  that 
animals  which  have  died  of  the  disease  have  been  buried  too 
near  the  surface.  It  is  therefore  advisable  that  animals  dead 
from  anthrax  be  buried  at  a  depth  not  less  than  six  feet  from 
the  surface,  as  the  soil  at  that  depth  is  15°  C.  even  in  sum- 
mer. Consequently  the  bacilli  developed  in  the  dead  bodies 
so  buried,  both  on  account  of  the  low  temperature  of  the  soil 
and  of  the  deprivation  of  oxygen,  will  not  form  spores  and 
are  not  likely  therefore  to  survive  for  any  length  of  time. 


DIPHTHERIA  AND  PSEUDODIPHTHERIA.  133 

QUESTIONS. 

Where  and  by  whom  was  the  Bacillus  anthracis  first  discovered  ? 

What  are  the  three  postulates  of  Koch  ? 

Describe  the  anthrax  bacillus? 

How  does  it  stain? 

How  does  it  appear  in  the  blood  of  animals?    How  in  culture-media  ? 

When  does  it  form  spores  ? 

How  does  it  grow  on  gelatin  ?    How  on  agar  ?    How  on  potato  ? 

At  what  temperature  does  it  grow  ? 

When  does  it  cease  to  form  spores? 

Are  spores  found  in  the  animal  body? 

How  resistant  are  the  spores  ? 

In  what  four  ways  are  animals  injected? 

How  are  animals  inoculated  ? 

Describe  tne  lesions  found  in  animals  after  subcutaneous  inoculation  ? 

How  are  cultures  attenuated  to  prepare  the  anthrax  vaccine? 

What  is  vaccine  No.  1  ?     Vaccine  No.  2  ? 

How  is  protecting  vaccination  practised  ? 


CHAPTER    XIV. 

DIPHTHERIA   AND  PSEUDODIPHTHERIA. 
DIPHTHERIA. 

Bacillus  Diphtherias. 

History. — The  infectious  nature  of  diphtheria  had  been  sus- 
pected for  a  long  time  when  Klebs  in  1883,  and  later  Loeffler 
in  1884,  discovered  and  accurately  described  in  the  false 
membranes  of  diphtheritic  patients  the  presence  of  a  micro- 
organism which  bears  their  combined  name — Klebs-Loeffler. 
Indeed,  no  infectious  disease  has  been  better  studied  from  its 
etiological  and  therapeutical  standpoints  than  diphtheria,  and 
it  conforms  absolutely  to  the  postulates  of  Koch  before  men- 
tioned :  that  is,  it  is  found  in  animals  sick  with  the  disease, 
it  may  be  cultivated  artificially,  and  pure  cultures  inoculated 
into  susceptible  animals  produce  the  disease.  The  disease  is 
not  produced  by  any  other  germs,  and  besides  injection  of  its 
toxins  produces  in  animals  substances  which  are  of  immu- 
nizing value  when  injected  into  susceptible  animals. 


134 


DIPHTHERIA   AND  PSEUDODIPHTHERIA. 


The  Bacillus  diphtheria  is  found  a.  in  false  membranes  of 
diphtheritic  origin  ;  6.  occasionally  in  the  mouth  and  nose  of 
healthy  individuals ;  and  c.  in  the  dust  of  rooms  inhabited 
by  diphtheritic  patients,  or  on  articles  of  clothing  or  furniture 
which,  though  they  may  not  have  come  into  direct  contact 
with  the  patients,  yet  have  been  in  the  same  room  with  them. 

Morphology. — The  Klebs-Loeffler  bacillus  is  a  short  rod, 
from  2  to  6  mikrons  in  length,  and  from  0.2  to  0.8  mikron 
in  breadth,  being  found  longer  in  certain  cultures  than  in 
others,  and  when  grown  for  several  generations  in  artificial 
media.  The  rods  occur  singly  or  in  pairs,  or  in  irregu- 
lar groups ;  they  may  be  straight  or  sometimes  slightly 
curved.  Occasionally  one  or  both  of  the  extremities  are 
thicker  than  the  rest  of  the  body  of  the  cell  ;  at  other  times 
the  centre  of  the  cell  bulges  and  the  end  of  the  cell  tapers 
(Figs.  56,  57,  58). 


FIG.  56. 


FIG.  57. 


One  of  very  characteristic  forms  of 
diphtheria  bacilli  from  blood-serum 
cultures,  showing  clubbed  ends  and  ir- 
regular stain.  X  1100.  Stain,  meth- 
ylene-blue.  (Park.) 


Extremely  long  form  of  diphtheria 
bacillus.  This  culture  has  grown  on 
artificial  media  for  four  years  and  pro- 
duces strong  toxin.  X  1100.  (Park.) 


Bacillus  diphtherice  stains  with  all  of  the  anilin  dyes  and 
by  Gram's  method,  but  better  with  Loeffler's  alkaline  meth- 
ylene-blue  solution.  For  the  purpose  of  differentiation  the 
Neisser  special  stain  is  often  used. 

The  bacilli  cells  do  not  stain  uniformly ;  they  contain  large 


DIPHTHERIA.  135 

granules,  occasionally  situated  at  one  or  both  extremities  or  in 
its  central  portion,  which  stain  much  more  deeply  than  the  rest 
of  the  cells,  and  which  make  of  a  stained  diphtheria  prepara- 
tion quite  a  characteristic  picture  under  the  microscope. 

Neisser's  Differential  Method. — Some  forms  of  false  diph- 
theria bacilli   which  can   not  be  separated  from    diphtheria 

FIG.  58. 


Diphtheria  bacilli  characteristic  in  shape  but  showing  even  staining.     In  appear- 
ance similar  to  the  xerosis  bacillus.    X  1100.    Stain,  methylene-blue. 

bacilli  by  their  mode  of  growth  or  by  their  appearance  under 
the  microscope,  but  which  are  not  toxic,  must  be  differen- 
tiated from  the  toxin-producing  bacilli  ;  and  Neisser  has 
suggested  the  following  method,  which  is  used  in  a  number 
of  municipal  laboratories.  It  consists  of  two  solutions,  as 
follows  : 

Solution  No.  1. 

Alcohol  (96  per  cent.),  20  parts ; 

Methylene-blue,  1  part ; 

Distilled  water,  950  parts  ; 

Glacial  acetic  acid,  50  parts  j 

Solution  No.  2. 

Bismarck -brown,  1  part ; 

Hot  distilled  water,  500  parts. 


136  DIPHTHERIA  AND  PSEUDODIPHTHERIA. 

Put  a  cover-glass  prepared  in  the  usual  way  for  two  or  three 
seconds  into  No.  1  ;  then  pass  into  No.  2  and  let  it  remain 
there  for  three  to  five  seconds ;  wash,  air-dry,  mount  in 
balsam.  The  body  of  the  bacteria  will  be  stained  brown, 
and  the  usually  darkly  stained  granules  with  the  Loeffler 
method  will  be  stained  blue.  If  the  bacilli  under  examina- 
tion are  true  diphtheria  bacilli,  the  majority  of  them  will  show 
the  blue  granule.  If  the  bacilli  are  pseudodiphtheritic  bacilli, 
scarcely  any  or  few  will  show  a  blue  stain  in  their  interior. 

Biologic  Characters. — The  Bacillus  diphtherice  is  aerobic, 
but  can  grow  in  the  presence  of  oxygen,  and  is  therefore  a 
facultative  anaerobic  ;  it  is  non-motile,  has  no  nagella,  does 
not  form  spores,  and  does  not  liquefy  gelatin. 

Its  thermal  death-point  is  58°  C.  It  grows  at  ordinary 
room  temperature,  but  slowly.  Its  maximum  of  growth  is 
between  37°  and  38°  C.  It  is  easily  killed  by  disinfectants. 
Exposure  to  direct  sunlight  destroys  the  bacilli  in  a  few 
days.  In  albuminous  fluid  and  in  the  dark  it  may  live,  even 
when  dried,  for  months.  It  grows  on  all  artificial  culture- 
media,  but  best  in  blood-serum  prepared  after  the  formula  of 
Loeffler,  a  modification  of  which,  employed  in  many  munic- 
ipal laboratories,  is  as  follows .: 

Blood -serum  from  sheep  or  calves,         3  parts  ; 
Peptone-bouillon  containing  1  per  cent. 

of  glucose,  1  part. 

Mix,  distribute  among  test-tubes,  sterilize,  and  harden  by  ex- 
posing in  a  slanting  position  in  a  steam  sterilizer  at  97°  C. 
for  two  hours. 

On  this  mixture  at  37°  C.  after  twelve  hours  the  colonies 
are  round,  grayish-white,  about  the  size  of  a  pin-head  ; 
later  they  become  larger,  elevated,  and  yellowish,  with  the 
centre  more  opaque  than  the  periphery.  At  the  end  of  a 
few  days  the  colonies  have  a  diameter  of  from  3  to  5  milli- 
meters. 

In  bouillon  at  37°  C.  the  cultures  present  small  clots 
deposited  on  the  side  and  at  the  bottom  of  the  tube.  Some 


DIPHTHERIA.  137 

of  the  culture  floats  on  the  surface  of  the  liquid,  forming  a 
thin  whitish  pellicle.  The  bouillon,  which  is  at  first  cloudy, 
becomes  in  a  few  days  clear,  and  remains  so.  The  sugars  con- 
tained in  the  bouillon  are  fermented,  and  it  is  due  to  their 
fermentation  that  this  medium  has  at  first  a  tendency  to  be 
acid  ;  but  subsequently,  when  the  fermentation  is  complete, 
become  decidedly  more  alkaline.  On  gelatin  the  colonies 
develop  very  slowly.  They  appear  white,  round,  irregu- 
larly notched,  and  somewhat  granular,  never  attaining  a  large 
size.  On  agar  the  growth  presents  the  same  characteristics 
as  on  blood-serum  ;  but  on  the  surface  of  agar  plates  the 
colonies  are  quite  characteristic,  having  a  dark  elevated  centre 
and  flat  periphery,  with  a  radiated  appearance  and  indented 
edges.  On  potato  the  growth  is  invisible  at  first ;  and  at 
the  end  of  several  days  a  thin  whitish  veil  seems  to  cover  the 
portion  of  the  potato  which  has  been  inoculated.  In  milk  it 
grows  at  a  temperature  as  low  as  20°  C.,  without  any  appre- 
ciable change  of  the  medium. 

Pathogenesis. — Diphtheria,  along  with  tetanus,  should  be 
classified  among  the  toxic  diseases.  As  a  matter  of  fact,  the 
symptoms  met  with  in  cases  of  diphtheria  are  due  to  the 
effects  of  the  toxins  secreted  by  the  bacilli;  very  few,  if  any, 
of  the  microorganisms  are  ever  found  in  the  blood  or  deep- 
seated  organs  in  cases  of  this  disease  ;  and  filtered  cultures  from 
which  the  bacilli  have  been  completely  eliminated,  when  inocu- 
lated into  animals  give  rise  to  symptoms  identical  with  those 
induced  by  inoculation  of  the  virulent  bacilli  themselves. 
Roux  and  Yersin,  by  the  filtration  of  cultures  through  un- 
glazed  porcelain,  have  been  able  to  separate  from  the  bacilli 
a  toxalbumin  which,  when  injected  under  the  skin  of  rabbits 
and  guinea-pigs,  produces  the  blood-poisoning,  renal  and 
nervous  symptoms  met  with  in  pure  diphtheria.  Welch  and 
Abbott  have  repeated  these  experiments,  and  having  estab- 
lished the  same  facts  have  come  to  the  same  conclusion  as  to 
the  action  of  this  toxalbumin. 

Subcutaneous  inoculations  of  the  diphtheria  bacilli  will  pro- 
duce death  in  guinea-pigs  in  about  thirty-six  hours.  The  fol- 
lowing lesions  are  found  at  the  autopsy  :  General  oedema  at 


138  DIPHTHERIA   AND  PSEUDODIPHTHERIA. 

the  point  of  inoculation,  with  the  formation  of  a  false  mem- 
brane. Marked  congestion  of  the  adrenal  bodies,  serous  or 
serosanguinolent  effusions  in  the  pleural  cavities,  and  swollen 
spleen.  A  few  of  the  bacilli  may  be  found  at  the  point  of 
inoculation  and  in  the  fluid  of  the  oedema.  In  the  blood  and 
internal  organs  no  bacilli  can  be  found,  showing  that  the 
symptoms  are  purely  toxic. 

Roux  and  Yersin  have  also  been  able  to  produce  the  false 
membrane  giving  rise  to  the  disease,  by  the  inoculation  of 
rabbits  and  guinea-pigs  into  the  mucous  surfaces  or  into  the 
skin,  and  they  have  reproduced  in  animals  the  characteristic 
diphtheria  paralysis.  This  paralysis,  best  seen  in  the  rabbit, 
usually  begins  in  the  posterior  extremities  and  gradually  ex- 
tends over  the  whole  body,  death  being  caused  by  paralysis 
of  the  heart  and  respiratory  organs. 

Different  cultures  of  diphtheria  bacilli,  though  emanating 
from  equally  virulent  cases  of  diphtheria,  and  grown  under 
the  same  conditions,  show  at  times  a  great  variation  in  toxicity. 
The  explanation  of  this  has  not  been  as  yet  satisfactorily 
given.  But  this  fact  we  should  remember  when  testing  the 
efficacy  of  antitoxins  in  neutralizing  the  toxins  of  diphtheria. 

Diphtheria  Diagnosis. — Clinically  it  is  not  always  easy  to 
differentiate  diphtheria  in  its  early  stages  from  other  affections 
of  the  throat  and  nose  which  are  characterized  by  the  pres- 
ence of  exudates.  In  view  of  the  recent  therapeutical  advances 
in  diphtheria,  it  is  important  that  a  very  early  diagnosis  be 
made.  For  this  purpose,  accepting  the  almost  unanimous 
opinions  of  experts,  that  diphtheria  is  due  to  the  presence  of 
diphtheria  bacilli  in  the  membranous  exudate,  boards  of 
health,  cities,  and  hospitals  have  established  a  diphtheria 
service  for  the  purpose  of  facilitating  the  early  recognition 
of  the  disease.  In  order  to  carry  out  this  method,  a  central 
laboratory  with  all  facilities  is  established,  and  in  cities  a 
number  of  supply-depots  are  located  within  reach  of  the 
practising  physician,  where  the  material  in  complete  outfits 
necessary  to  make  cultures  from  the  throats  of  suspected  cases 
of  diphtheria  may  be  procured.  These  outfits  consist  of  a  blood- 
serum  culture-tube  (Fig.  59)  made  after  the  formula  of  Loeffler, 


DIPHTHERIA.  139 

and  a  swab  or  applicator  kept  in  a  well-sterilized  test-tube. 
This  swab  is  a  small  iron  rod  roughened  on  one  of  its  ends, 
and  on  which  a  little  absorbent  cotton  is  twisted.  The  test- 
tube  containing  the  swab  is  plugged  with  absorbent  cotton  and 
then  thoroughly  sterilized  by  dry  heat  for  one  hour  at  150°  C. 
The  blood-serum  and  swab  are  neatly  packed  together  in  a 
small  pasteboard  or  wooden  box,  together  with  a  blank  form 
giving  instructions  as  to  how  to  make  the  cultures. 
The  cultures  from  the  throat  are  made  as  follows : 
The  patient  is  put  in  the  best  possible  light,  and  if  he  is  a 
child  is  held  firmly  by  an  assistant,  the  mouth  is  opened,  the 

FIG.  59. 


Culture-box  used  in  municipal  laboratories  to  prepare  cultures  from  throats  of 
diphtheria  suspects. 

tongue  depressed  by  means  of  a  spoon  or  other  instrument, 
the  swab  taken  out  of  its  containing  tube  and  gently  rubbed 
over  the  false  membrane  or  exudate  in  the  throat,  if  any, 
or  if  no  false  membrane  be  present,  over  the  surface  of  the 
pillar  of  the  fauces,  after  which,  without  laying  down  the 
swab,  the  serum-tube  is  taken,  the  plug  of  cotton  removed, 
and  the  surface  of  the  swab  which  has  been  in  contact  with 
the  throat  of  the  patient  is  gently  and  freely  rubbed  over  the 
surface  of  the  blood-serum,  being  careful  not  to  break  into  it. 


140  DIPHTHERIA  AND  PSEUDOD1PHTHERIA. 

and  certain  to  rub  all  sides  of  the  swab  upon  the  serum. 
After  which  the  swab  is  returned  to  its  tube,  both  tubes 
plugged,  and  the  whole  outfit  with  the  blank  form  filled  in  is 
returned  to  the  laboratory.  On  receiving  the  tube  at  the 
laboratory  it  is  incubated  at  a  temperature  of  37°  C.  for 
twelve  hours,  at  the  end  of  which  time  it  is  ready  for  exami- 
nation. If  the  case  is  one  of  diphtheria,  the  typical  diph- 
theria growth  is  found  on  the  surface  of  the  culture.  This 
consists  of  grayish  or  yellowish-white  glistening  spots,  and  a 
cover-glass  preparation  made  of  these  shows  in  typical  cases 
the  Klebs-Loeffler  bacillus,  as  short,  thick  rods,  with  rounded 
edges,  irregular  in  shape,  showing  a  decided  staining  in  some 
parts  of  their  body,  deficient  in  color  in  other  parts,  and 
characterized  chiefly  by  the  variety  of  form  of  the  different 
bacteria  forming  the  culture. 

In  exceptional  cases  it  is  possible  to  find  colonies  as  early 
as  five  or  six  hours  after  incubation. 

Indeed,  for  cases  outside  of  the  city  limits,  in  the  munic- 
ipal laboratory  in  New  Orleans,  it  has  been  possible  to  make 
examinations  of  the  swabs  themselves  by  making  cover-glass 
preparations  from  the  same  even  two  or  three  days  after  they 
were  prepared,  and  in  a  great  majority  of  the  cases  come  to 
a  positive  or  negative  conclusion,  verified  later  clinically  and 
also  bacteriologically,  by  cultures  made  from  these  same 
swabs. 

It  is  essential  for  these  examinations  that  the  cultures  from 
the  throats  of  suspected  cases  be  made  before  antiseptics  have 
been  applied  to  the  throat,  or,  if  that  is  not  possible,  the  cult- 
ures should  be  made  at  an  interval  of  at  least  two  or  three 
hours  after  such  applications,  as  otherwise  the  antiseptics 
may  have  acted  on  the  bacilli  on  the  surface  of  the  membrane 
and  destroyed  them  or  greatly  inhibited  their  growth. 

PSEUDODIPHTHERIA. 
Bacillus  Pseudodiphtherise. 

Another  source  of  error  in  the  application  of  this  method 
comes  from  the  pseudodiphtheria  bacilli  which  are  found  in 


PSEUDODIPHTHERIA.  141 

cultures,  and  which  greatly  resemble  the  virulent  Bacillus 
(liji/itheriw,  but  have  no  pathogenic  power. 
These  pseudobacilli  are  of  two  kinds : 

I.  It  is  not  possible  to  separate  the   first  kind  from  the 
true   diphtheria   bacilli    either    by   morphology    or   cultural 
properties.     When  injected  into  the  lower  animals  they  are 
non-virulent,  because  they  secrete  no  toxin. 

II.  The  second  kind,  in  the  opinion   of  the  author,   are 
very  improperly  so-called,  for  they  are  not  diphtheria  bacilli, 
and  can  with  little  difficulty  be  differentiated  from  true  diph- 
theria bacilli  by  their  appearance,  mode  of  staining,  and  their 
cultural  properties. 

Differential  Diagnosis. — The  method  of  staining  suggested  by 
Neisser,  as  mentioned  in  the  beginning  of  the  chapter,  is 
applicable  especially  to  the  recognition  of  the  second  form  of 
pseudobacilli. 

For  the  recognition  of  the  non-toxin-producing  form,  ex- 
periment on  animals  is  the  only  means  of  differentiating. 

What  appear  to  be  true  diphtheria  bacilli  have  been  found 
in  the  throat  and  mouth  in  about  1  per  cent,  of  a  number 
of  healthy  persons  examined,  but  generally  in  individuals 
who  have  come  into  contact  with  diphtheria  patients,  or  when 
diphtheria  was  prevalent  in  the  community  at  the  time  of 
the  examination.  Those  persons  are  always  a  source  of 
danger  to  others,  and  they  no  doubt  are  in  a  great  measure 
responsible  for  the  spread  of  the  disease. 

The  experiments  of  Roux  and  Yersin  have  shown  that  the 
various  cultures  of  diphtheria  bacilli  have  different  potency 
in  the  production  of  toxins,  and  that  occasionally  bacilli 
grown  under  conditions,  the  same  as  much  as  possible,  may  at 
different  times  produce  more  or  less  toxins,  and  of  a  greater 
or  lesser  virulence.  These  facts  bacteriologists  are  in  no 
position  to  explain,  and  the  toxicity  of  a  diphtheria  culture 
may  only  be  determined  by  experimentation  on  animals. 

The  Antitoxin  Treatment  of  Diphtheria. 

The  discovery  made  by  Roux,  that  the  diphtheria  bacilli 
secrete  a  toxin  which,  when  injected  into  susceptible  ani- 


142  DIPHTHERIA  AND  PSEUDODIPHTHERIA. 

mals,  produces  all  the  symptoms  of  true  diphtheria,  was 
soon  followed  by  the  discovery  of  Behring,  which  showed  that 
the  blood-serum  of  animals  injected  with  the  bacilli  of  diph- 
theria contains  a  substance  which  when  inoculated  into  sus- 
ceptible animals  is  able  to  immunize  them  from  lethal  doses 
of  the  bacilli. 

These  substances,  called  antitoxins,  are  obtained  from  ani- 
mals having  little  or  no  susceptibility  to  the  disease,  and 
they  have  been  used  extensively  both  in  the  prevention  and 
cure  of  diphtheria  since  1894. 

These  antitoxins  as  exhibited  therapeutically  are  obtained 
from  the  blood-serum  of  horses,  as  first  suggested  by  Roux, 
and  are  prepared  as  follows  : 

Immunization. — A  good-sized  horse,  which  has  been  demon- 
strated to  befree  from  tuberculosis  and  glanders,  by  the  injecting 
of  tuberculin  and  mallein,  and  free  from  all  rheumatic  and  chronic 
disease,  is  gradually  immunized  to  the  diphtheritic  poison  by 
being  injected  with  very  small  doses  of  the  virulent  toxins 
from  a  diphtheria  bouillon  culture  filtrated  through  porcelain. 
The  initial  dose  consists  of  0.10  c.c.  mixed  with  an  equal 
quantity  of  Gram's  iodine  solution ;  this  should  produce 
little  or  no  constitutional  disturbance,  and  very  little  if  any 
local  effect.  Four  or  five  days  after  this  first  injection  a 
second  injection,  consisting  of  pure  toxin  0.10  c.c.,  is  used, 
and  every  four  or  five  days  thereafter  injections  are  re- 
peated in  progressively  larger  doses  until  the  animal  is 
able  to  withstand  doses  of  from  400  to  500  c.c.  of  toxin. 
During  those  injections  the  animal  may  show  decided  local 
effects,  such  as  swelling  and  osdema  at  the  point  of  inocula- 
tion, but  no  very  marked  constitutional  disturbances.  During 
the  progress  of  this  immunization,  at  intervals,  by  punctur- 
ing of  the  jugular  vein  with  a  sterilized  trocar,  some  blood 
is  withdrawn  from  the  animal  and  its  serum  tested  as  to  its 
antitoxic  value,  and  when  the  same  is  found  sufficient  the 
toxin  injections  are  repeated  at  longer  intervals  to  maintain 
the  antitoxic  property  of  the  animal's  serum,  and  the  next 
process  is  begun. 

Standardization. — A  large  quantity  of  blood,  4  or  5  liters, 


PSEUDODIPHTHERIA.  143 

is  extracted  from  the  immunized  horse  at  one  time,  collected 
in  well-sterilized  vessels,  and  allowed  to  clot  in  an  ice-chest 
for  two  or  three  days,  after  which  the  clear  serum  is  pipetted 
off  and  stored  in  sterilized  flasks,  the  antiseptic  strength  of 
the  serum  being  properly  labelled  on  each  flask.  This  anti- 
V  toxin  power,  called  units,  is  estimated  as  follows : 

Ten  times  a  fatal  dose  of  a  toxin,  that  is  known  to  kill  a 
^-250-gram  guinea-pig  within  three  days,  is  mixed  with  differ- 
ent quantities  of  the  serum  to  be  tested,  say,  0.10,  0.01, 
0.001  c.c.,  and  these  mixtures  injected  into  different  guinea- 
pigs,  Nos.  1,  2,  and  3,  respectively.  Should  guinea-pig  No.  1 
survive  the  mixed  injection,  and  guinea-pigs  Nos.  2  and  3 
die,  the  antitoxin  is  said  to  contain  10  times  10  units  in  1  c.c. ; 
that  is,  it  is  an  antitoxin  of  100-unit  power.  Should  guinea- 
pigs  1  and  2  survive,  the  antitoxin  is  one  which  in  1  c.c.  has 
protecting  powers  amounting  to  10  multiplied  by  20,  or  200 
antitoxin  units.  Should  guinea-pig  No  3  also  survive  this 
injection,  then  the  serum  used  is  equivalent  to  10  times  100, 
or  1000  antitoxin  units  per  c.c. 

No  serum  should  be  accepted  for  use  in  the  treatment  of  diph- 
theria unless  its  immunizing  or  antitoxic  power  is  equivalent  to 
at  least  WO  units  per  c.c.;  a  serum  used  as  a  protective  only 
may  be  accepted  with  100  units  antitoxic  power  per  c.c. 

In  order  to  test  the  antitoxin  and  for  the  purpose  of  im- 
munizing animals,  it  is  necessary  to  produce  toxins  of  a 
standard  virulence.  This,  as  has  been  seen,  is  not  always  a 
task  of  easy  performance.  The  standard  of  toxins  accepted 
in  all  laboratories  and  establishments  in  which  antitoxin  is 
manufactured  is  a  toxin  of  which  0.10  c.c.  is  able  to  kill  a 
250-  or  300-gram  guinea-pig  within  three  days,  and  no  toxins 
should  be  used  excepting  such  as  have  this  power.  It  is  best 
manufactured  by  growing  virulent  cultures  of  Bacillus  diph- 
therias in  large  Erlenmeyer  flasks,  with  free  access  of  air  and 
at  a  temperature  of  37°  C.  The  height  of  the  toxicity  of 
the  culture  is  reached  in  about  eight  to  ten  days,  when  the 
culture  should  be  removed  from  the  incubator  and  filtered 
through  a  Chamberlain  porcelain  filter,  tested  on  guinea-pigs, 
and  if  found  of  the  required  strength  put  away  in  sterile 


144  DIPHTHERIA  AND  PSEUDO DIPHTHERIA. 

bottles.  Unless  it  shows  that  0.10  c.c.  when  injected  into 
a  guinea-pig  of  250  grams  causes  death  of  the  animal  within 
three  days,  it  should  not  be  accepted. 

The  German  government  adopted  this  as  a  standard  strength 
for  toxins,  and  no  antitoxin  is  put  on  the  market  unless  its 
value  has  been  tested  by  means  of  its  power  of  neutralizing 
so  many  units  of  this  standard  toxin. 

Value  of  the  Antitoxin  Treatment  of  Diphtheria. — It  has  now 
been  used  eight  years ;  and  has  been  of  inestimable  impor- 
tance. As  a  therapeutic  agent  given  within  the  first  three  days 
of  the  disease,  it  has  reduced  the  mortality  of  diphtheria 
more  than  one-half.  When  used  after  the  third  day  it  is  of 
less  value,  but  still  shows  decidedly  good  effects. 

When  used  as  a  preventative  in  persons  exposed  to  the 
danger  of  contagion  with  this  disease,  it  gives  protection  for 
several  weeks. 

Dose  of  Antitoxic  Serum. — As  a  prophylactic  from  200  to 
500  units  should  be  used,  according  to  the  age.  For  the 
purpose  of  treatment  not  less  than  2000  to  3000  units  should 
be  injected  at  one  time,  and  that  as  early  as  possible  in  the 
course  of  the  disease  ;  and  this  dose  should  be  repeated  in 
twenty-four  hours  unless  decided  beneficial  effects  are  noticed. 

The  experience  of  the  author,  based  on  the  examination 
of  several  thousand  cases  of  diphtheria  treated  by  serum  in 
New  Orleans,  has  shown  that,  with  the  exception  of  an  occa- 
sional urticarial  rash,  no  untoward  effect  follows  this  treat- 
ment. The  explanation  of  this  eruption  has  not  been  given, 
but  it  is  very  probably  due  to  some  other  elements  contained 
in  the  blood -serum  of  the  horse,  and  appears  to  be  much 
more  common  following  the  use  of  serum  taken  from  some 
horses  than  from  that  of  others ;  it  appears  to  have  no  rela- 
tion to  the  antitoxic  power  of  the  serum. 

QUESTIONS. 

When  and  by  whom  was  the  Bacillus  diphtherix  discovered  ? 
How  does  it  answer  the  postulates  of  Koch  with  regard  to  pathogenic 
bacteria  ? 

Where  is  the  Bacillus  diphtherise  found  ? 

Describe  the  appearance  of  the  Klebs-Loeffler  bacillus. 


TETANUS,  MALIGNANT  (EDEMA,  ETC.  145 

Describe  the  staining  of  this  bacteria. 

What  characterizes  cultures  of  the  diphtheria  bacillus? 

How  are  false  or  pseudobacilli  differentiated  from  true  diphtheria  bacilli? 

Describe  the  Neisser  method  of  staining  the  diphtheria  bacilli. 

How  does  it  behave  in  the  presence  of  oxygen? 

Is  it  motile  ? 

Has  it  flagella  ? 

Does  it  contain  spores? 

At  what  temperature  does  it  grow? 

What  is  its  thermal  death-point  ? 

How  does  it  behave  in  the  presence  of  disinfectants? 

How  is  it  affected  by  direct  sunlight? 

How  does  it  behave  in  the  albuminous  fluid?    How  in  the  dark? 

How  is  Loefller's  blood-serum  for  the  culture  of  the  Bacillus  diphtherise 
prepared  ? 

Describe  the  growth  of  this  bacillus  on  Loefller's  medium,  in  bouillon,  on 
gelatin,  on  agar,  on  potato,  in  milk? 

Why  is  diphtheria  a  toxic  disease? 

How  is  the  toxin  of  diphtheria  obtained? 

Give  the  effects  of  inoculation  of  diphtheria  bacilli  on  guinea-pigs. 

What  is  the  effect  of  the  inoculation  of  those  bacteria  on  mucous  surfaces 
of  animals? 

How  do  the  different  cultures  of  Bacillus  diphtherise  vary  as  to  their 
virulence  ? 

Give  the  boards'  of  health  measures  for  diagnosing  diphtheria  by  means 
of  cultures. 

How  is  the  inoculation  of  cultures  made  in  those  cases? 

What  are  the  sources  of  error  in  this  form  of  examination? 

What  two  forms  of  pseudobacilli  are  found  ? 

How  are  they  recognized  from  true  virulent  bacilli  ? 

How  is  the  toxin  prepared  ? 

How  is  it  gauged  ? 

What  is  constant  diphtheria  toxin? 

What  is  the  result  of  the  antitoxin  treatment  of  diphtheria? 

What  is  the  result  of  its  prophylactic  use  ? 

What  dose  should  be  given  as  a  prophylactic? 

What  dose  should  be  given  in  the  treatment  of  diphtheria  cases? 


CHAPTER    XV. 

TETANUS,    MALIGNANT    (EDEMA,     AND    SYMPTOMATIC 

ANTHRAX. 

TETANUS. 
Bacillus  Tetani. 

History. — Bacillus  tetani  was  discovered  by  Nicolaier  in  1884, 
and  cultivated  by  Kitasato  in  1889. 
10— M.  B. 


146 


TETANUS,  MALIGNANT  (EDEMA,  ETC. 


It  is  found  a.  in  wounds  in  cases  of  tetanus,  b.  as  a  sapro- 
phyte in  the  soil,  especially  manured  soil  of  gardens  and 
stables,  and  c.  in  the  intestinal  secretions  of  animals. 

Morphology. — The  bacillus  of  tetanus  as  obtained  in  cult- 
ures is  seen  in  one  of  two  forms,  either  in  the  vegetative  form 
or  as  a  spore -bearing  bacterium. 

Its  vegetative  form  is  a  short  rod  with  round  ends,  occur- 
ring singly  or  in  pairs,  or  sometimes  forming  long  filaments. 

FIG.  60. 


Bacillus  tetani :  A,  vegetative  stage ;  B,  spore-stage,  showing  pin-shapes.    (Abbott.) 

Its  spore-bearing  form  is  quite  characteristic,  resembling  a 
pin  ;  this  is  due  to  the  fact  that  the  spore  is  formed  at  one 
end  of  the  bacillus,  and  as  the  bacillus  bulges  at  that  portion 
the  typical  appearance  of  a  pin  is  given  to  the  bacillus 
(Fig. '60).  _ 

The  Bacillus  tetani  stains  with  all  the  anilin  dyes,  and  also 
by  Gram's  method. 

Biologic  Characters. — The  Bacillus  tetani  is  purely  an- 
aerobic, not  developing  at  all  in  the  presence  of  oxygen.  It 
grows  in  all  culture-media  at  a  temperature  as  low  as  18°  or 


TETANUS. 


147 


FIG.  61. 


20°  C.,  but  best  at  37°  C.  It  does  not  grow  at  a  tempera- 
ture below  14°  C.  Cultures  must  be  kept  in  a  hydrogen 
atmosphere,  as  the  presence  of  the  oxy- 
gen of  the  air  prevents  their  growth. 

On  the  surface  of  gelatin  the  cultures 
resemble  very  much  those  of  the  Ba- 
cillus subtilis,  but  liquefy  the  medium 
more  slowly  (Fig.  61). 

In  gelatin  stab-cultures  it  grows  in 
the  depth  of  the  medium,  and  the  col- 
onies have  very  much  the  appearance 
of  a  fir  tree.  Its  growth  is  very  slow 
in  this  medium,  but  the  addition  of 
from  1  to  2  per  cent,  of  glucose  to  the 
gelatin  increases  materially  the  rapidity 
of  the  growth.  The  growth  on  agar  is 
very  much  like  that  on  gelatin,  but  it 
causes  no  liquefaction  of  the  medium. 

In  bouillon  it  grows  at  37°  C.,  in  the 
depth  of  the  tube,  with  the  production 
of  gases.  It  does  not  cause  coagula- 
tion of  milk,  and  produces  no  acids  in 
its  cultures.  All  cultures  of  it  are  noted 
for  their  characteristic  disagreeable  odor. 

To  obtain  pure  cultures  of  the  Bacillus 
tetani,  a  number  of  methods  have  been 
resorted  to  ;  that  recommended  by  Kit- 
asato  is  as  follows : 

The  pus  or  secretion  of  the  wound  in 
a  case  of  tetanus,  or  some  garden  or 
stable  soil  containing  the  sporing  form 
of  Bacillus  tetani,  is  plated  on  agar,  or 
streak  cultures  are  made  on  this  me- 
dium witli  the  secretions  of  the  wound 
or  with  the  contaminated  soil.     These 
agar  plates  or  tubes  arc  kept  at  the     fraenkei  and  p'feiffer.) 
temperature  of  the  incubator  for  three  or  four  days,  so  as 
to  allow  the  growth  of  all  bacteria  contained  therein.     At 


Colonies  of  the  tetanus 
bacillus  four  days  old.made 
by  distributing  the  organ- 
is'ms  through  a  tube  nearly 
filled  with  glucose-gelatin. 
Cultivation  in  an  atmos- 
phere of  hydrogen.  (From 


148  TETANUS,  MALIGNANT  (EDEMA,  ETC. 

the  end  of  that  time  cover-glass  preparations  are  made 
from  the  colonies  along  the  streak  or  on  the  surface  of  the 
agar  plates.  If  some  of  the  characteristic  pin-shaped  Ba- 
cillus tetani  are  found,  the  cultures  are  treated  as  follows  : 
They  are  exposed  from  three-fourths  to  one  hour  to  the 
temperature  of  80°  C. ;  in  this  way  all  the  fully  formed 
bacteria  and  the  greater  part  of  the  spores  are  killed,  the 
vegetative  form  of  the  Bacillus  tetani  included,  but  spores  of 
this  bacillus  remain  alive.  Then  from  these  cultures  fresh 
gelatin,  bouillon,  or  agar  tubes  are  inoculated,  and  the  same 
grown  at  the  temperature  of  the  room  or  incubator  in  an 
atmosphere  of  hydrogen.  If  the  original  substance  experi- 
mented with  contained  the  Bacillus  tetani,  characteristic  cult- 
ures will  be  seen  in  this  medium  in  a  few  days,  and  may 
subsequently  be  transplanted. 

Motility  and  Thermal  Death-Points. — The  Bacillus  tetani  in 
the  spore-bearing  variety  is  non-motile  ;  the  vegetative  form  is 
quite  motile,  though  no  flagella  have  been  discovered.  A 
temperature  of  58°  C.  will  destroy  the  non-spore-bearing 
variety  in  a  half-hour ;  60°  C.  will  kill  them  in  five 
minutes  ;  and  65°  C.  instantaneously.  Spores,  however,  are 
able  to  resist  a  temperature  of  80°  C.  for  two  hours,  but  are 
killed  by  a  temperature  of  100°  C.  in  from  four  to  five  min- 
utes. When  dried,  the  spores  are  capable  of  retaining  their 
vitality  for  months  and  years.  Carbolic  acid  (5  per  cent.) 
will  not  kill  them  in  less  than  ten  hours  ;  but  if  0.5  per 
cent,  hydrochloric  acid  be  added  to  the  carbolic  acid  solution, 
spores  will  be  destroyed  in  two  hours.  Bichloride  of  mer- 
cury (1  in  1000)  will  destroy  them  in  three  hours.  Bi- 
chloride (1  in  1000)  to  which  6.5  per  cent,  hydrochloric  acid 
has  been  added  will  kill  them  in  thirty  minutes. 

Tetanin. — The  Bacillus  tetani  secretes  a  powerful  poison, 
known  as  tetanin,  which  diffuses  in  the  cultures  and  is  not 
retained  in  the  cell-body. 

The  symptoms  of  tetanus  are  due  to  the  action  of  this 
toxin,  and  not  to  the  influence  of  the  bacteria  themselves. 
Tetanus  is  strictly  a  toxsemic  disease.  This  is  proved  by  the 
fact  that  inoculations  with  cultures  of  bacilli  in  which  the 


TETANUS.  149 

toxins  have  been  destroyed  produce  no  symptoms  whatever. 
These  toxins  are  destroyed  by  a  temperature  of  60°  to  65°  C., 
by  prolonged  exposure  to  diffuse  daylight,  or  by  exposure  for 
one  hour  to  direct  sunlight,  and  cultures  containing  spores 
so  exposed  are  innocuous  to  animals. 

The  author  has  succeeded  in  several  instances  in  obtaining 
the  tetanus  bacillus  by  the  following  cultivation-method  : 

After  thoroughly  heating  a  bouillon  tube  or  a  liquid  gela- 
tin tube  so  as  to  expel  as  much  as  possible  all  the  oxygen, 
the  culture  is  allowed  to  cool  to  a  temperature  a  little  below 
80°  C.  The  suspected  material  is  then  inoculated  deep  into 
the  tube,  and  the  surface  of  the  medium  is  covered  by  a  layer 
of  1  to  2  c.c.  of  paraffin  oil,  a  cotton  plug  inserted,  and  a 
rubber  cap  applied  over  the  tube. 

In  this  way  he  has  obtained  cultures  with  great  facility.  In 
one  case,  notably,  cultures  were  made  from  the  surface  of  a 
nail,  that  caused  a  wound  which  produced  tetanus  in  an  adult. 
In  another  case  a  piece  of  diphtheritic  membrane  wrapped  in  a 
piece  of  gauze  and  kept  on  the  hearth  over  night,  was  handed 
to  him  from  a  diphtheria  patient  for  examination.  The  next 
day  to  his  surprise  besides  the  diphtheria  bacilli  a  few  bacilli 
resembling  the  tetanus  bacilli  were  found.  A  piece  of  this 
membrane  was  inoculated  into  bouillon  prepared  in  the  fore- 
going manner,  and  he  obtained  after  three  or  four  days  a  pure 
culture  of  the  tetanus  bacillus  which  proved  fatal  to  guinea- 
pigs. 

Pathogenesis. — The  animals  susceptible  to  the  Bacillus  tetani 
are  man,  horses,  guinea-pigs,  rabbits,  and  mice.  Dogs  are 
little  susceptible,  and  birds  scarcely  at  all.  Amphibians  can 
not  be  infected.  The  inoculation  of  animals  is  made  by  means 
of  a  liquid  culture  injected  subcutaneously  or  by  means  of 
some  of  the  contaminated  material  introduced  into  a  deep 
pocket  in  the  subcutaneous  tissue.  The  period  of  incubation 
is  more  or  less  prolonged,  varying  from  a  few  days  to  occasion- 
ally two  or  three  weeks.  During  this  time  the  bacilli  seem  to 
be  generating  their  poison.  After  this  has  been  accomplished, 
the  toxic  effects  are  very  marked  and  rapidly  fatal,  the  symp- 
toms showing  first  in  the  parts  nearest  to  the  point  of  inocu- 


150  TETANUS,  MALIGNANT  (EDEMA,  ETC. 

lation.  These  symptoms  consist  in  spasms  of  the  muscular 
system,  and  generally  end  in  death.  The  blood  and  the  urine 
of  inoculated  animals  is  toxic  to  other  susceptible  animals. 

At  the  autopsy,  apart  from  the  slight  inflammatory  changes 
at  the  point  of  inoculation,  with  occasionally  the  discovery  of 
a  few  bacilli  at  that  point,  no  changes  are  observed  in  the 
organs,  excepting  an  intense  congestion  of  the  nervous 
system. 

Bacilli  deprived  of  toxins  injected  into  animals  are  taken  up 
by  the  phagocytes. 

Preparation  of  the  tetanus  toxin  is  very  easy.  A  bouillon 
culture  of  the  Bacillus  tetani  is  grown  in  an  atmosphere  of 
hydrogen  at  a  temperature  of  37°  C.  for  from  two  to  four 
weeks.  At  the  end  of  that  time  the  culture  is  filtered 
through  a  porcelain  filter,  and  the  filtrate  is  found  to  contain 
the  tetanin,  which  is  best  kept  in  the  dark,  and  preserved  by 
the  addition  of  0.5  per  cent,  of  phenol.  The  power  of  this 
toxin,  called  tetanin,  is  very  great,  5-0  oliTo-o  c'c*  ^eing  suffi- 
cient to  kill  a  15-gram  mouse  in  three  to  four  days.  Occa- 
sionally this  toxicity  is  very  much  increased,  and  Burger  and 
Cohn  have  succeeded  in  obtaining  tetanin,  which  in  doses  of 
c-c'  was  fetal  to  mice.  This  is  b  far  the  most 


FoTTooT  o 

powerful  poison  known;   taken   in   this   proportion  it  would 

mean  that  about  ^  milligram  would  be  fatal  to  man.  Com- 
pare this  with  atropine,  the  fatal  dose  of  which  is  about  130 
milligrams,  and  anhydrous  prussic  acid,  the  fatal  dose  of 
which  is  54  milligrams,  and  a  fair  idea  of  its  toxicity  will  be 
obtained. 

Tetanin  acts  on  animals  only  when  introduced  into  the  cir- 
culation; given  by  the  mouth  it  possesses  no  poisonous  prop- 
erties. 

The  blood  of  animals  dead  or  affected  with  tetanus  is  poi- 
sonous to  other  animals  in  the  same  way  as  cultures  of  the 
bacillus  itself.  But  it  is  possible  to  inoculate  animals  with 
doses  small  enough  to  produce  no  fatal  effects  ;  and  animals 
so  inoculated  are  protected  from  future  infection,  and  their 
blood  and  fluid  secretions  will  serve  to  protect  other  animals 
when  injected  in  doses  less  than  the  fatal  dose.  The  dis- 


TETANUS.  151 

covery  of  this  fact  by  Behring  and  Kitasato  has  been  the  open- 
ing wedge  to  serum  therapy. 

Tetanus  antitoxin,  like  diphtheria  antitoxin,  is  produced  by 
inoculating  large  animals,  like  the  horse,  with  minute  doses 
of  the  toxin,  diluted  at  first  with  Gram's  iodine  solution,  and 
artificially  establishing  in  the  horse  an  immunity  against  the 
poison.  The  dose  of  the  toxin  is  gradually  increased,  and 
injected  every  few  days  into  the  animals  until  immense  doses 
(600  to  700  c.c.)  may  be  injected  at  one  time  without  pro- 
ducing any  marked  symptoms.  When  immunity  has  thus 
been  secured,  blood  is  taken  from  the  animal  and  its  serum 
tested,  when  it  is  found  to  have  decided  powers  of  neutralizing 
the  toxin. 

Tetanus  antitoxin  is  useful  chiefly  as  a  preventative  against 
tetanus,  and  in  veterinary  medicine  has  been  found  of  great 
value.  When  applied  to  the  human  subject,  however,  the 
results  have  not  been  so  satisfactory,  for  it  is  used  then  only 
as  a  therapeutic  agent.  At  the  time  of  its  employment  the 
symptoms  of  tetanus  have  generally  shown  themselves,  and 
these  are  exceedingly  rapid  and  violent  in  their  effects,  and 
commonly  fatal.  A  number  of  observers  have  derived  very 
decided  benefit  from  its  use,  however,  especially  by  injecting 
it  into  the  ventricles  of  the  brain,  where  it  may  act  by  directly 
and  locally  combating  the  poisonous  action  of  the  tetanin 
present. 

Tetanus  antitoxin  is  measured  somewhat  differently  than  is 
diphtheria  antitoxin.  Its  strength  is  expressed  as  follows  : 
1  in  1,000,000  or  I  in  10,000,000.  This  means  that  1  c.c. 
of  the  antitoxin  is  capable  of  protecting  from  infection 
1,000,000  or  10,000,000  grams  of  guinea-pig.  In  some 
cases  an  antitoxin  of  800,000,000-gram  power  has  been 
obtained. 

This  antitoxin,  however,  does  not  retain  its  power  very 
long,  and  deteriorates  quickly  in  the  fluid  form.  It  is  gener- 
ally made  into  a  powder,  which  may  be  dissolved  into  a 
neutral  saline  solution  for  use. 


152  TETANUS,  MALIGNANT  (EDEMA,  ETC. 

MALIGNANT  (EDEMA. 
The  Bacillus  of  Malignant  (Edema. 

History. — Malignant  oedema  is  caused  by  a  very  malignant 
bacillus,  discovered  by  Pasteur,  studied  by  Koch  and  Kitt,  and 
found  in  the  soil  of  gardens  and  in  the  dust  of  streets,  which, 
when  inoculated  into  animals,  rapidly  produces  the  disease. 

Morphology. — Bods  from  3  to  5  mikrons  in  length  and  1.10 
mikron  in  thickness.  They  occur  singly  or  in  pairs  in  cult- 
ures, rarely  forming  threads.  The  ends  are  square  in  appo- 
sition when  two  bacilli  come  together,  but  rounded  when  the 
bacilli  are  single  or  at  the  free  ends  of  united  bacilli. 

This  bacillus  stains  with  all  the  ordinary  methods  of  stain- 
ing, but  does  not  stain  by  Gram's  method. 

It  forms  spores,  situated  at  or  near  the  centre  of  the  ba- 
cillus, causing  a  swelling  of  the  bacterium.  (Plate  III.) 

Biologic  Characters. — The  bacillus  of  malignant  cedema  is 
an  obligate  anaerobic,  and  does  not  grow  at  all  in  the  presence 
of  oxygen.  It  grows  in  all  culture-media  in  hydrogen  gas, 
liquefies  gelatin,  and  rapidly  liquefies  blood-serum. 

In  gelatin  and  bouillon  it  grows  at  the  bottom  of  the  tube, 
and  in  the  liquid  gelatin  the  colonies  are  in  the  form  of 
spheres,  which  are  scarcely  discernible  at  first,  but  which,  on 
account  of  the  fermentation  developed  by  the  bacilli  causing 
clouding  of  the  medium,  become  more  and  more  apparent. 

On  agar  plates  in  a  hydrogen  atmosphere  it  grows  as  whit- 
ish bodies,  which  under  the  magnifying  glass  are  seen  to 
consist  of  branching  and  interlacing  lines  radiating  irregu- 
larly from  the  centre  to  the  periphery. 

The  colonies  grow  at  ordinary  temperature,  but  best  at 
37°  C. 

Pathogenesis. — Men,  horses,  calves,  dogs,  sheep,  chickens, 
pigeons,  rabbits,  guinea-pigs,  are  all  susceptible  to  the 
disease. 

Inoculation  of  animals  is  performed  subcutaneously  by  in- 
troducing a  small  particle  of  the  suspected  material  or  culture 
into  a  deep  pocket.  The  symptoms  developed  in  animals  are 
a  rapid  and  extensive  oedema,  with  bloody  effusions  at  the 


PLATE  III 


Bacillus  CEdematis  Maligni.     (Abbott.) 

A.  (Edema-fluid,  from  site  of  inoculation  of  guinea-pig,  showing  long  and 
short  threads.     B.  Spore-formation,  from  culture. 


SYMPTOMATIC  ANTHRAX.  153 

point  of  inoculation,  involving  also  the  muscular  tissues. 
The  internal  organs  show  little  change,  excepting  the  spleen, 
which  is  enlarged.  The  bacilli  are  rarely  found  in  the  blood 
of  the  heart  when  the  autopsy  is  performed  immediately 
after  death,  but  they  are  found  in  limited  numbers  in  the 
internal  viscera.  If  the  autopsy  is  delayed,  however,  the 
whole  body  of  the  animal  becomes  infected  with  the  bacillus. 
This  bacillus  is  grown,  like  the  tetanus  and  other  anaerobics, 
in  atmospheres  of  hydrogen  only. 

SYMPTOMATIC  ANTHRAX. 
Bacillus  Anthracis  Symptomatici. 

History. — Ferrer  and  Bellinger  discovered  a  bacillus  in  the 
disease  of  animals  known  as  black  leg,  quarter  evil,  or  quarter 
ill,  which  is  also  found  in  humid  soils  in  certain  localities 
during  the  summer  months,  especially  when  those  places  have 
been  contaminated  with  discharges  from  infected  animals. 

Morphology. — The  description  of  this  microorganism  given 
by  Kitasato  is  as  follows  : 

Actively  motile  rods,  3  to  5  mikrons  in  length,  and  from 
0.5  to  0.6  mikron  in  thickness,  occurring  singly,  occasionally 
in  pairs,  never  forming  filaments  (Fig.  62). 

It  stains  by  all  the  anilin  colors  and  by  Gram's  method. 
It  forms  spores,  which  are  situated  at  or  near  one  of  the  poles, 
giving  a  swollen  appearance  to  the  bacillus. 

Biologic  Characters. — In  the  vegetative  type  it  is  actively 
motile,  but  loses  its  motion  in  the  spore-bearing  form.  It 
can  not  be  cultivated  in  an  atmosphere  of  oxygen.  It  is 
purely  anaerobic,  and  does  not  grow  in  an  atmosphere  of  car- 
bonic acid  gas. 

It  grows  best  when  glucose  (1.5  to  2  per  cent.)  or  glyc- 
erin (4  to  5  per  cent.)  is  added  to  the  culture-medium.  It 
grows  in  all  media.  It  liquefies  gelatin.  It  grows  best 
at  the  temperature  of  the  incubator,  37°  C.,  but  does  not 
grow  at  a  temperature  below  14°  C.  In  deep-seated  punct- 
ures of  gelatin  or  agar  it  grows  in  three  or  four  days,  and 
produces  during  its  growth  gas  bubbles.  The  colonies  appear 


154 


TETANUS,  MALIGNANT  (EDEMA,  ETC. 


as  globules  which  cause  liquefaction  of  the  gelatin  and 
coalesce  into  irregular  tabulated  liquid  areas.  The  dried 
spores  retain  their  vitality  for  months.  They  resist  a  tem- 
perature of  80°  C.  for  one  hour,  but  five  minutes'  exposure 
at  100°  G.  is  sufficient  to  destroy  them.  Carbolic  acid 
(5  per  cent.)  is  not  effective  as  a  disinfectant  in  less  than  ten 
hours.  The  vegetative  form,  however,  is  killed  in  from  three 

FIG.  62. 


Bacillus  of  symptomatic  anthrax:  A,  vegetative  stage— gelatin  culture; 
B,  spore-forms — agar-agar  culture.    (Abbott.) 

to  five  minutes.     Bichloride  of  mercury  (1  :  1000)   will  kill 
the  spores  in  two  hours. 

Pathogenesis. — Cattle,  sheep,  goats,  guinea-pigs,  and  mice 
are  susceptible  animals.  Horses,  asses,  and  rats  show  only 
slight  local  swelling,  but  no  general  infection.  Dogs,  cats, 
rabbits,  chickens,  pigeons,  and  hogs  are  immune.  Inocula- 
tions are  generally  made  deep  into  the  subcutaneous  tissue 
either  with  pure  cultures  of  the  microorganisms  or  from  bits 
of  tissue  of  a  suspected  animal.  The  symptoms  are  a  rise  of 
temperature,  followed  by  painful  swelling  at  the  point  of 
inoculation.  Death  takes  place  in  from  one  to  two  days. 


QUESTIONS.  155 

The  autopsy  reveals  an  extensive  swelling  of  the  subcutane- 
ous tissues  with  emphysema.  The  oedematous  fluid  is  blood- 
stained, and  the  muscles  are  dark  and  prominent.  The 
lymphatic  glands  are  involved.  The  internal  organs  show 
little  change.  In  the  fluid  of  the  oedema  the  bacilli  are  found 
in  large  numbers,  lying  singly.  Early  autopsy  reveals  no 
bacteria  in  the  blood,  only  a  few  in  the  internal  organs. 
Late  autopsy  shows  a  considerable  quantity  of  organisms  that 
have  invaded  the  whole  body.  The  bacilli  in  the  body  arc- 
found  to  contain  spores.  This  serves  as  a  differentiation, 
in  addition  to  other  points,  between  it  and  the  Bacillus 
anthraeis. 

Immunity. — One  attack  of  the  disease  if  not  fatal  affords 
protection  against  future  attacks.  The  opposite  of  this  hap- 
pens with  malignant  oedema,  one  attack  of  which  seems  to 
predispose  to  other  attacks. 

QUESTIONS. 

When,  where,  and  by  whom  was  the  Bacillus  tetani  discovered  and  culti- 
vated? 

How  many  forms  of  the  Bacillus  tetani  are  there,  and  how  are  these  dis- 
tinguished ? 

What  is  the  characteristic  appearance  of  the  spore-bearing  form  ? 

In  what  atmosphere  does  it  grow  best,  and  why? 

What  is  the  temperature-limit  of  its  growth  ? 

How  does  it  grow  in  gelatin ?    In  agar?    In  bouillon?    In  milk? 

What  is  Kitasato's  method  of  obtaining  pure  cultures  of  this  bacillus? 

In  what  form  is  it  motile  ? 

What  agents  and  chemicals  are  the  spores  capable  of  resisting,  and  to  what 
extent? 

What  is  tetanin? 

Describe  a  method  of  growing  anaerobic  bacilli  with  the  use  of  paraffin  oil. 

What  animals  are  susceptible  to  the  infection  of  tetanus? 

Describe  the  autopsy  of  an  animal  inoculated  with  Bacillus  tetani. 

Where  and  how  are  inoculations  in  animals  made? 

What  symptoms  are  produced  by  tetanin  injection? 

What  type  of  infection  is  tetanus  ? 

How  is  tetanus  toxin  prepared? 

What  is  the  degree  of  toxieity  of  tetanin? 

How  is  the  antitoxin  of  tetanus  prepared? 

How  is  it  used  and  for  what  purpose? 

Why  is  it  of  more  use  in  veterinary  than  in  human  medicine? 

How  is  the  strength  of  tetanus  antitoxin  expressed? 

By  whom  was  discovered  the  bacillus  of  malignant  oedema? 

Where  is  it  found  ? 

What  is  its  appearance  ? 


156  TYPHOID  FEVER. 

How  does  it  stain  ? 

How  does  it  behave  in  the  presence  of  oxygen? 

How  does  it  grorv  on  different  media  ? 

What  animals  are  susceptible  ? 

How  are  inoculations  performed  ? 

What  symptoms  a  e  produced  by  inoculation? 

By  whom  was  tho  bacillus  of  symptomatic  anthrax  discovered? 

Where  is  it  found  ? 

What  diseases  of  animals  are  produced  by  it? 

Give  the  description  of  microorganisms  containing  spores  and  vegetative 
forms. 

How  does  it  stain  ? 

What  effect  does  the  addition  of  glucose  to  media  have  upon  the  growth 
of  this  organism  ? 

How  does  it  grow  in  different  media  ? 

What  are  the  effects  of  temperature  on  its  growth  ? 

How  is  it  fatal  to  animals  ? 

What  animals  are  susceptible? 

How  is  it  differentiated  from  other  bacilli  ? 

What  is  the  effect  of  a  non-fatal  attack  of  this  disease? 

How  does  symptomatic  anthrax  compare  with  malignant  oedema? 


CHAPTER    XYI. 

TYPHOID  FEVER. 

Bacillus  Typhosus. 

History. — The  presence  of  a  microorganism  in  cases  of 
typhoid  fever  was  discovered  by  Eberth,  in  1880;  it  was 
named  the  Bacillus  typhosus  ;  but  until  isolated  and  described 
by  Gaffky,  in  1884,  it  was  not  fully  recognized. 

It  is  found  after  death  in  the  blood,  spleen,  liver,  intestines, 
Peyer's  patches,  and  mesenteric  ganglia,  and  during  life  in  the 
blood,  especially  when  the  same  is  taken  from  the  spleen  by 
means  of  a  hypodermatic  syringe,  in  the  rose  patches,  in  the 
urine  and  feces,  and  outside  the  human  body,  occasionally  in 
water  and  soil  contaminated  with  dejecta  of  typhoid  patients, 
and  often  in  milk,  which  is  due  probably  to  the  cleansing  of 
the  utensils  in  which  the  milk  is  collected  with  water  con- 
taminated with  the  bacilli  (Figs.  63  and  64). 

Morphology. — The  Bacillus  typhosus  appears  as  a  rod  with 


BACILLUS  TYPHOSUS. 


157 


rounded  extremities,  from  2  to  4  mikrons  in  length,  and  0.6  to 
0.8  mikron  in  breadth.  At  times  it  appears  as  short  ovals  ;  at 
others  the  bacilli  are  joined  together,  forming  long  threads. 
It  stains  with  all  the  anilin  dyes,  but  not  quite  so  readily  as 
other  bacteria.  It  does  not  stain  by  Gram's  method.  In 
stained  preparations  clear  spaces  are  observed  in  the  body  of 
the  cells.  This  has  given  rise  to  the  belief  that  the  bacteria 
contain  spores.  There  are,  however,  no  spores,  for  those  clear 
spaces  do  not  stain  by  any  of  the  spore-staining  processes,  and 
bacteria  in  which  they  are  found  are  less  resistant  to  external 


FIG.  63. 


FIG.  64. 


/', 


Wtof 


Bacillus  typhosus,  from  culture 
twenty-four  hours  old,  on  agar- 
agar.  (Abbott.) 


Bacillus  typhosus,  showing  flagella 
stained  by  Loeffler's  method.  (Abbott.) 


influences  than  others.  This  bacillus  has  numerous  fine,  hair- 
like  flagella,  which  are  not  to  be  seen  in  unstained  prepara- 
tions or  preparations  stained  by  the  ordinary  methods,  but 
it  requires  the  flagella-stain  of  Loeffler  to  bring  them  out. 

Biologic  Characters.  —  The  Bacillus  typhosus  is  aerobic,  but 
grows  also  without  the  presence  of  oxygen  ;  it  is  therefore 
facultative  anaerobic.  It  is  non-spore-bearing,  and  is  actively 
motile,  the  motions  at  times  being  very  rapid.  It  grows  in 
nearly  all  the  artificial  media,  even  at  the  room  temperature, 
but  best  at  a  temperature  of  37°  C.  Its  growth  at  20°  C.  is 
rather  slow,  but  quite  rapid  at  the  temperature  of  the  body. 

On  gelatin  plates  its  colonies  appear  as  small,  yellowish, 
punctiform  bodies,  becoming  in  a  short  time  round  and 


158  TYPHOID  FEVER. 

irregularly  notched,  resembling  droplets  of  oil.  In  gelatin 
stab-cultures  they  appear  as  small  thick  disks,  finely  dentated, 
of  a  pearl-like  color.  They  do  not  liquefy  gelatin. 

On  agar  plates  they  appear  as  round,  irregular,  shiny  colo- 
nies of  a  blue  or  grayish-white  color,  and  develop  very  abun- 
dantly. In  agar  stab-cultures  the  growth  is  chiefly  on  the 
surface,  and  in  the  depth  of  the  medium  there  is  scarcely  any 
appreciable  development. 

On  lactose-litmus  agar  colonies  are  pale  blue.  On  potato 
the  growth  is  exceedingly  variable,  and  not  characteristic,  as 
formerly  believed.  Sometimes  it  is  scarcely  appreciable,  at 
other  times  it  forms  a  film  like  a  thin  veil  of  the  same  color 
as  the  potato  itself.  Again,  at  times  the  growth  is  somewhat 
luxuriant  and  of  a  whitish  color.  It  does  not  coagulate  milk. 
It  does  not  cause  fermentation  in  glucose-,  lactose-,  or  sac- 
charose-bouillon. It  does  not  produce  indol  in  such  quantity  as 
is  detected  by  the  ordinary  tests. 

Vitality. — It  is  killed  by  an  exposure  of  ten  minutes  to 
60°  C.,  and  in  much  shorter  time  by  exposure  to  higher  tem- 
peratures. In  the  dried  conditions  it  may  be  preserved  for 
months. 

Agglutination. — Persons  who  have  suffered  from  an  attack 
of  typhoid  fever  or  animals  which  have  been  inoculated  with 
cultures  of  this  bacillus  have  generated  in  their  blood-serum 
a  substance  called  agglutinin.  This  agglutinin  has  the  prop- 
erty when  mixed  with  cultures  of  the  Bacillus  typhosus  of 
suddenly  arresting  the  motion  of  the  bacilli  and  of  causing 
their  clumping  or  agglutination,  which  is  quite  characteristic, 
and  is  made  use  of  for  the  diagnosis  of  typhoid  fever,  as 
will  be  described  later. 

Pathogenesis. — None  of  the  lower  animals,  as  far  as  has 
been  ascertained,  is  naturally  susceptible  to  contract  or 
develop  typhoid  fever.  Indeed,  the  typical  lesions  of  the 
disease  as  found  in  man  have  rarely  been  induced  in  the 
lower  animals  by  inoculations  with  the  typhoid  bacillus. 

Intraperitoneal,  subcutaneous,  and  intravascular  inoculations, 
in  rabbits,  guinea-pigs,  and  mice,  will  produce  marked  infec- 
tion even  in  those  animals,  in  the  form  of  general  septicaemia, 


DIFFERENTIATION  OF  THE  BACILLUS  TYPHOSUS.   159 

in  which  the  bacilli  have  been  recovered  in  the  general  cir- 
culation and  in  the  internal  organs.  The  feeding  of  animals 
with  articles  contaminated  with  typhoid  fever  germs  has  in 
some  instances,  when  the  animal's  vitality  was  very  much 
lowered,  produced  infection,  and  sometimes  lesions  in  the 
intestines  and  mesenteric  ganglia  very  much  resembling 
those  found  in  human  beings. 

Differentiation  of  Bacillus  Typhosus  from  Allied 
Groups. 

I.  General  Features. — In  many  respects  the  Bacillus  typho- 
sus  resembles   very  much  the  Bacillus   coli   communis,  both 
from  a  morphological  point  of  view  as  well  as  in  its  cultural 
peculiarities.     The  differentiation  between  the  two  is  some- 
times quite  difficult,  and  it  is  necessary  to  cultivate  the  bacilli 
in  all  the   known   artificial  media  to  come  to  a  conclusion 
about  their  identity.     The  points  of  differentiation  are  the 
following : 

The  Bacillus  coli  communis  is  generally  thicker  and  much 
less  motile  than  the  typhoid  bacillus.  The  coli  communis 
grows  much  more  rapidly  in  all  media.  The  flagella  of  the 
typhoid  bacillus  are  more  numerous.  The  Bacillus  typhosus 
does  not  coagulate  milk,  and  the  coli  communis  does.  Its 
growth  on  litmus-agar  remains  blue,  that  of  the  coli  com- 
munis becomes  red  from  the  production  of  acids.  The  Ba- 
cillus typhosus  produces  no  indol,  as  ascertained  by  the  ordinary 
Dunham's  test,  but  the  coli  communis  produces  indol  very 
rapidly.  The  Bacillus  typhosus  does  not  produce  fermenta- 
tion in  lactose  or  glucose  media,  whereas  the  coli  communis 
produces  fermentation  and  fermentative  gases.  On  potato 
the  growth  of  Bacillus  typhosus  is  almost  invisible,  while  that 
of  Bacillus  coli  communis  is  abundant,  creamy,  and  of  a  dark- 
brown  color.  The  serum  of  the  blood  from  typhoid  fever 
cases  agglutinates  cultures  of  Bacillus  typhosus.  It  has  no 
action  on  Bacillus  coli  communis 

II.  Widal's  and  Chantemesse's  Differentiation. — Two  tubes 
of  agar  or  gelatin  to  which  2  per  cent,  of  lactose-sugar  has 


160  TYPHOID  FEVER. 

been  added  are  allowed  to  melt  and  a  sufficient  quantity  of 
neutral  litmus  tincture  is  added  to  them  to  give  a  deep- 
violet  color.  The  tubes  are  sterilized  and  are  inoculated, 
one  with  the  Bacillus  typhosus,  and  the  other  with  the  Ba- 
cillus coli  communis.  If  agar  tubes  are  used,  they-  are  placed 
in  the  incubator  at  37°  C.  When  the  colonies  grow,  those 
of  the  Bacillus  typhosus  retain  the  blue  color,  while  the 
colonies  of  the  Bacillus  coli  communis  become  of  a  bright-red 
color,  and  at  the  bottom  of  the  tube  can  be  seen  bubbles  of  gas. 

III.  Eisner's  Method  of  Differentiation. — This  consists  in 
employing  an  acid  mixture  of  gelatin,  potato  juice,  and  potas- 
sium iodide,  which  contains  neither  peptone  nor  sodium  chlo- 
ride. It  is  used  to  separate  not  only  the  coli  communis,  but 
also  the  ordinary  saprophytes  from  the  Bacillus  typhosus. 
The  saprophytes  do  not  develop  at  all  in  this  medium,  and 
the  colonies  of  coli  communis  and  Bacillus  typhosus  show 
marked  differences  in  their  behavior  on  plates  made  of  this 
mixture,  and  are  easily  separated.  At  the  end  of  twenty- 
four  hours  tubes  of  this  mixture  inoculated  with  the  sus- 
pected material  will  contain  a  large  number  of  coli  communis 
colonies,  which  have  the  same  appearance  as  cultures  of  this 
bacillus  on  ordinary  agar  plates,  whereas  there  will  scarcely 
be  visible  development  of  colonies  of  the  Bacillus  typhosus. 
After  forty-eight  hours  the  Bacillus  typhosus  will  appear  as 
small,  pale,  almost  transparent  colonies,  easily  distinguished 
from  the  dark  granular  colonies  of  the  coli  bacillus.  Accord- 
ing to  Abbott,  Eisner's  medium  is  thus  prepared : 

"Grate  1  kilogram  of  pealed  potato  and  allow  this  to  stand 
over  night  in  a  refrigerator ;  then  press  out  all  juice,  using 
an  ordinary  meat-press  for  the  purpose  ;  filter  this  fresh  juice 
cold  to  remove  as  much  of  the  starch-granules  as  possible. 
If  this  is  not  done,  the  starch  when  heated  swells  to  such  an 
extent  as  to  render  filtration  almost  impracticable.  Boil  the 
filtrate  and  again  filter.  Test  the  filtrate  for  acidity  by 
titrating  10  c.c.  with  a  decinormal  solution  of  sodium  hy- 
droxide, the  indicator  used  being  6  drops  of  the  ordinary 
0.5  per  cent,  solution  of  phenolphtalein  in  50  per  cent, 
alcohol.  The  acidity  of  the  juice  should  be  such  as  to  re- 


DIFFERENTIATION  OF  THE  BACILLUS  TYPHOSUS.   161 

quire  3  c.c.  of  a  decinormal  sodium  hydroxide  solution  to 
neutralize  10  c.c.  of  the  juice.  If  the  acidity  is  found  to  be 
greater  than  this,  which  is  usually  the  case,  dilute  with  water 
until  the  proper  degree  is  reached.  If  less  than  this,  the 
juice  may  be  concentrated  by  evaporation.  It  is  desirable 
that  this  acidity  should  be  due  to  the  acids  normally  present 
in  the  potato,  and  that  it  should  not  be  artificially  obtained 
by  the  addition  of  other  acids.  Now  add  10  per  cent,  of 
gelatin  (with  no  peptone  and  no  sodium  chloride  present), 
dissolve  by  boiling,  and  again  test  the  acidity,  using  10  c.c. 
of  the  mixture  and  phenolphtalein  as  before.  Deduct  3  c.c. 
(the  acidity  of  the  potato  juice  that  is  to  be  maintained)  from 
the  number  of  c.c.  of  the  decinormal  sodium  hydroxide  solu- 
tion requisite  to  neutralize  the  10  c.c.  of  the  gelatin  mixture, 
and  from  the  resulting  figure  calculate  the  amount  of  normal 
solution  of  sodium  hydroxide  needed  for  the  entire  volume, 
and  add  it.  Boil,  clarify  with  an  egg,  and  filter  through 
paper  in  the  usual  manner.  To  the  filtrate  add  potassium 
iodide  in  the  proportion  of  1  per  cent.,  decant  into  tubes,  and 
sterilize." 

IV.  Stodard's  and  Hiss'  Differentiation. — By  this  method 
use  is  made  of  the  great  motility  of  the  Bacillus  typhosus  to 
differentiate  it  from  the  coli  communis.  It  is  valuable  at 
times.  Success  in  this  procedure  depends  on  the  important 
fact  that  in  a  semifluid  mixture  the  Bacillus  typhosus,  on 
account  of  its  great  motility,  will  diffuse  much  more  rap- 
idly from  the  point  of  inoculation  to  nearly  all  parts  of  the 
medium,  whereas  the  coli  communis,  having  only  a  sluggish 
or  no  motion  at  all,  develops  only  at  the  place  of  immediate 
inoculation.  For  detailed  accounts  of  these  methods  the 
reader  is  referred  to  larger  treatises  on  bacteriology. 

Sources  of  Pure  Cultures. — From  the  spleen  of  typhoid 
fever  cases  pure  cultures  of  the  bacillus  may  be  readily  ob- 
tained, in  early  autopsies,  and  during  life  ;  blood  extracted 
by  means  of  a  hypodermatic  syringe  from  this  organ  will 
almost  always  show  the  bacillus.  Indiscriminate  punctures 
of  the  spleen  during  life,  however,  are  not  to  be  recom- 
mended, as  this  procedure  is  not  free  from  danger. 
11— M.  B. 


162  TYPHOID  FEVER. 

The  Bacillus  typkosus  has  occasionally  been  obtained  froni 
abscesses  in  the  subcutaneous  tissue  and  internal  organs  in 
pure  cultures  in  some  cases  of  typhoid  fever,  showing  that 
this  bacillus  is  at  times  the  cause  of  suppuration. 

Artificial  Susceptibility. — Animals  resisting  the  effects  of 
inoculation  with  the  Bacillus  typhosus  can  be  made  suscepti- 
ble by  the  simultaneous  introduction  of  other  saprophytes 
which  seem  to  overcome  their  immunity. 

The  Blood-Serum  Diagnosis  of  Typhoid  Fever. 

The  diagnosis  of  typhoid  fever  by  the  blood-serum  method 
is  to-day  generally  employed.  As  mentioned  before,  this  is 
based  on  the  principle  discovered  by  Pfeiffer,  that  the  blood  of 
persons  suffering  with  typhoid  fever,  or  who  may  recently 
have  had  the  disease,  when  mixed  with  young  cultures  of 
Eberth's  bacillus,  has  the  property  of  arresting  the  active 
motion  of  the  bacilli,  and  causing  their  agglutination  or 
clumping.  This  power  resides  in  the  serum,  and  is  due  to  a 
substance  called  agglutinin. 

Widal  inaugurated  the  blood-test  for  typhoid  fever,  and  sug- 
gests that  to  1  c.c.  of  bouillon  culture,  not  more  than  twenty- 
four  hours  old,  and  grown  at  a  temperature  of  35°  C.,  0.10  c.c. 
of  the  serum  to  be.  tested  be  added.  The  serum  may  be 
obtained  either  by  allowing  the  drawn  blood  to  coagulate,  or 
by  means  of  a  small  blister.  In  the  space  of  from  five  to 
ten  minutes  all  motion  of  the  bacilli  is  arrested,  and  these 
come  together,  forming  peculiar  clumps.  This  clumping  may 
be  seen  both  in  the  hanging  drop,  and  even  by  the  naked  eye 
in  culture-tubes.  Ordinarily  the  hanging-drop  method  is 
adopted,  as  it  requires  much  less  serum,  and  is  therefore  less 
injurious  and  vexatious  to  the  patient, 

Wyatt  Johnston's  Dried  Blood  Method. — This  observer  has 
demonstrated  that  the  same  reaction  may  be  obtained  by  the 
use  of  dried  blood  instead  of  fresh  serum,  and  that  even  after 
the  blood  has  been  dried  for  several  days  or  weeks  it  still 
retains  its  agglutinating  power.  The  procedure  in  detail  is  as 
follows : 


THE  BLOOD-SERUM  DIAGNOSIS  OF  TYPHOID  FEVER.      163 

A  drop  of  the  blood  to  be  tested  is  obtained  from  the  finger 
or  lobe  of  the  ear  and  allowed  to  dry  on  a  clean  slide.  With 
a  platinum  wire  a  few  loopfuls  of  sterile  water  are  mixed  with 
the  dried  blood  and  the  same  is  diluted  until  about  of  the 
same  color  as  normal  blood.  One  loopful  of  this  blood  mixt- 
ure is  added  to  40  or  50  loopfuls  of  a  bouillon  culture  of 
the  Bacillus  typhosus  twenty  hours  old,  on  a  cover-glass,  and 
a  hanging  drop  made  in  the  usual  way.  In  the  course  of  a 
half-  to  one  hour,  if  the  blood  comes  from  a  case  of  typhoid 
fever  of  sufficient  duration,  not  less  than  six  or  seven  days, 
cessation  of  motion  and  clumping  of  the  bacteria  in  the 
culture  drop  will  have  been  completely  effected. 

In  the  experience  of  the  author  in  'the  Municipal  Labora- 
tory of  New  Orleans  with  more  than  6000  cases,  this  test 

FIG.  65. 


Outfit  used  by  the  Municipal  Laboratory  of  New  Orleans  for  the  collection 
of  blood  for  the  typhoid  fever  test. 

has  given  satisfactory  results.  The  plan,  which  is  a  modifica- 
tion of  the  New  York  Board  of  Health  method,  is  as  follows : 

At  the  diphtheria  depots  blood  slides  are  left  with  blank 
forms  giving  directions  (Fig.  65). 

Directions  for  Preparing  Specimen  of  Blood. — Clean  thor- 
oughly the  tip  of  the  finger  or  lobe  of  the  ear,  and  prick  with 
a  clean  needle  deep  enough  to  cause  several  drops  of  blood  to 
exude  ;  two  or  three  drops  are  then  placed  on  the  slide  of  the 
outfit.  Let  the  blood  dry,  then  place  the  slide  in  holder,  fill 
out  the  blank  form,  and  return  to  depot  where  obtained.  On 
the  following  day  a  report  of  the  result  of  examination  will 
be  mailed  or  telephoned  to  the  attending  physician. 


164  TYPHOID  FEVER. 

The  blood  only  of  fever  patients  is  to  be  used.  Should  the 
report  be  negatived  and  the  case  be  suspicious,  the  physician 
in  attendance  is  requested  to  send  another  specimen,  and  in 
every  case  to  notify  the  bacteriologist  as  to  whether  the  labo- 
ratory diagnosis  is  finally  in  harmony  with  the  clinical  diag- 
nosis or  at  variance  with  it. 

Sources  of  Error. — One,  which  must  be  remembered,  is  due 
in  some  cases  to  the  persistence  of  the  reaction  for  a  number 
of  years  after  a  typhoid  attack  :  so  that  a  reaction  may  appear 
in  health  or  in  affections  other  than  typhoid  fever,  if  the 
patient  has  previously  suffered  from  the  disease.  In  cases  in 
which  the  reaction  is  marked,  it  may  apparently  be  positively 
stated  that  the  patient  has,  or  has  had,  typhoid  fever  within 
a  few  years. 

Diagnostic  Values. — If  the  reaction  is  present,  but  not  well 
marked,  only  probable  diagnosis  may  be  made.  If  the  reac- 
tion is  absent  in  a  patient  sick  seven  days,  the  diagnosis  of 
typhoid  fever  may  be  excluded. 

The  experiment  has  not  been  tried  long  enough  and  not  in  a 
sufficient  number  of  cases  to  permit  a  positive  statement  as  to  the 
earliest  date  of  the  appearance  of  the  reaction  in  typhoid  fever. 

Vaccination  Against  Typhoid  Fever. 

Wright  and  Semple  have  recently  practised  the  vaccination 
of  human  beings  against  typhoid  fever,  and  extensive  ob- 
servations have  been  made  in  India  and  South  Africa  in  the 
British  Army.  For  this  purpose  a  typhoid  vaccine  consisting 
of  a  bouillon  emulsion  made  from  a  slant  agar  culture  of  the 
Bacillus  typhosus  twenty-four  hours  old  is  used.  The  cult- 
ure is  killed  by  heating  it  for  five  minutes  at  a  temperature 
of  60°  C.  From  a  half  to  a  quarter  of  the  whole  culture 
is  used  for  one  vaccination,  and  the  culture  must  be  of  such 
a  strength  that  a  fourth  of  it  is  capable  of  killing  a  300- 
to  400-gram  guinea-pig,  when  the  same  is  injected  into  it, 
without  killing  the  bacilli. 

The  results  obtained  by  these  vaccinations  have  been  en- 
couraging and  seem  to  open  up  a  promising  field  for  the 
serum-therapy  of  typhoid  fever. 


BACILLUS  COLI  COMMUNIS.  165 

Antityphoid  Serum. — Bokenham  has  recently  succeeded  in 
immunizing  a  horse  by  using  a  filtered  bouillon  culture  of  the 
typhoid  bacillus,  and  he  claims  that  the  horse's  serum  has 
immunizing  power  when  injected  into  guinea-pigs. 

QUESTIONS. 

What  name  is  usually  given  to  the  microorganism  causing  typhoid  fever? 

By  whom  and  when  was  it  discovered  ? 

Where  is  it  found  in  the  human  body? 

Where  is  it  occasionally  found  outside  of  the  human  body? 

Describe  the  Bacillus  typhosus. 

What  are  its  staining  peculiarities? 

How  do  you  stain  the  flagella  of  the  Bacillus  typhosus  ? 

Why  do  you  say  that  it  contains  no  spores? 

How  does  it  behave  in  the  presence  of  oxygen  ? 

Is  it  motile  ? 

At  what  temperature  does  it  grow  best  ? 

What  is  its  growth  on  gelatin?  On  agar?  On lactose-litmus-agar ?  On 
potato?  In  Dunham's  solution?  In  the  fermentation-tube ? 

Does  it  liquefy  gelatin  ? 

What  is  the  thermal  death-point  of  the  Bacillus  typhosus  ? 

What  is  agglutinin  ? 

How  are  animals  inoculated  with  the  Bacillus  typhosus  f 

What  are  the  points  of  difference  between  the  Bacillus  typhosus  and  the 
Bacillus  coli  communis  f 

Give  the  Widal-Chantemesse  method  of  distinguishing  between  colonies 
of  typhoid  and  coli  communis? 

Give  Eisner's  method  of  separating  the  Bacillus  typhosus  from  the  Bacillus 
coli  communis  and  water  bacteria. 

Give  Abbott's  mode  of  preparing  Eisner's  medium. 

On  what  are  Stodard's  and  Hiss'  methods  based? 

In  what  organ  may  the  bacillus  be  obtained  in  pure  cultures? 

How  may  the  resistance  of  animals  to  typhoid  inoculation  be  overcome? 

On  what  does  the  serum-test  of  typhoid  fever  depend  ? 

How  may  serum  be  obtained  for  this  test  ? 

Describe  the  methods  pursued  with  dried  blood  in  municipal  laboratories. 


CHAPTER    XVII. 

Bacillus  Coli  Gommunis. 

History. — It  was  discovered  by  Escherich,  1885,  and  is  found 
in  health  as  a  constant  inhabitant  of  the  intestinal  tract — 
chiefly  in  the  large  intestine — and  also  in  the  excretions  from 


166  BACILLUS  COLI  COMMUNIS. 

that  tract.  In  pathological  conditions  it  is  met  with,  in  asso- 
ciation with  other  bacteria,  a.  In  acute  enteritis,  cholera 
morbus,  in  certain  forms  of  dysentery  ;  it  is  easily  demon- 
strable in  large  numbers,  and  has  been  thought  by  some  to 
be  the  cause  of  those  diseases,  but  this  is  not  so.  Its  pres- 
ence in  the  healthy  individual  in  nearly  all  cases  is  sufficient 
to  show  the  falsity  of  this  position,  b.  It  has  also  been 
found  in  cases  of  peritonitis,  endocarditis,  and  in  suppurating 
inflammation  of  the  liver  and  the  kidney.  At  autopsies  it 
occurs  in  various  organs  and  in  nearly  all  conditions.  Asso- 
ciated with  specific  microorganisms  it  has  also  been  proved 
to  exist  in  the  blood  of  patients  in  articulo  mortis.  Outside 
the  human  body  it  has  been  discovered  in  water  and  soil  con- 
taminated with  fecal  matter. 

Etiologic  Relations. — For  a  long  time  this  bacillus  was 
looked  upon  as  a  harmless  saprophyte  ;  latterly  experiments 
have  established  the  fact  that  it  is  often  the  cause  of  inflam- 
matory conditions  in  the  body,  and  that  in  a  number  of  other 
instances  it  is  pathogenic  from  the  fact  that  it  lowers  the 
vitality  of  the  body  and  enables  other  germs  to  act  delete- 
riously. 

Morphology. — This  bacillus  is  polymorphous  and  very 
closely  resembles  the  typhoid  bacillus  in  shape.  It  is  a  rod 
with  rounded  extremities,  in  very  young  cultures  appearing 
almost  oval  with  a  bright  centre.  Later  on  the  bacilli  coa- 
lesce and  appear  as  long  threads.  They  possess  flagella  ;  not 
so  numerous,  however,  as  the  Bacillus  typhosus.  These  fla- 
gella may  be  stained  by  the  Loeffler  method.  It  has  no 
spores  and  stains  by  all  the  ordinary  anilin  dyes,  but  not  by 
the  Gram  method. 

Biologic  Characters. — It  is  aerobic  and  facultative  anaerobic. 
It  is  motile  at  times,  and  at  other  times  appears  to  be  motion- 
less. Its  motility  is  always  of  the  sluggish  kind.  Cultures 
which  when  young  contain  organisms  with  decided  motion, 
have  on  being  kept  for  some  time  shown  that  the  bacilli  have 
lost  their  motility.  It  grows  on  all  the  artificial  culture- 
media  and  at  the  temperature  between  10°  and  40°  C.  Its 
growth,  though  retarded  at  the  temperature  above  40°  C., 


BACILLUS  COLI  COMMUNIS.  167 

is  not  altogether  stopped  until  45°  C.  is  reached.  Exposure 
to  a  temperature  of  65°  C.  for  five  minutes  destroys  the  bac- 
teria. Exposure  to  cold  has  no  effect  on  the  bacteria,  and  in 
some  instances  the  author  has  been  able  to  cultivate  bacteria 
which  had  been  exposed  to  the  temperature  of  liquefied  air 
for  several  minutes. 

In  bouillon  the  bacillus  grows  very  rapidly  and  renders  the 
bouillon  cloudy  ;  pellicles  are  formed  on  the  surface  of  the 
medium,  and  there  is  also  a  thick  deposit  at  the  bottom  of 
the  tube.  A  strong  fecal  odor  can  be  detected. 

On  gelatin  plates  the  colonies  appear  as  small,  spherical, 
blue-gray  points,  somewhat  dentated  at  the  margin.  With  a 
magnifying  glass  the  colonies  are  brownish,  lozenge-shaped 
or  irregularly  round,  coarsely  granular.  In  gelatin  stab- 
cultures  along  the  track  of  the  needle  are  seen  a  series  of 
small  spherical  colonies  in  rows  and  separated  from  each  other. 
On  the  surface  of  the  tube  the  growth  is  of  a  dirty  gray 
color.  It  does  not  liquefy  gelatin. 

On  agar-agar  the  growth  has  nothing  characteristic.  On 
agar  to  which  2  per  cent,  glucose  has  been  added  bubbles  may 
be  seen  along  the  line  of  growth,  due  to  the  gases  of  fermen- 
tation. On  lactose-litmus-agar  the  colonies  develop  very 
rapidly  and  are  of  a  pinkish  color.  On  potato  it  grows  rap- 
idly in  the  beginning,  being  of  a  bright-yellow  color  which  later 
becomes  brown.  The  growth  in  serum  is  similar  to  that  on  agar. 

It  produces  indol  in  peptone  solution  and  coagulates  milk 
very  rapidly.  It  ferments  lactose-  and  glucose-bouillon. 

Pathogenesis. — Bouillon  cultures  of  this  bacillus  injected 
intravenously  or  into  the  peritoneal  cavity  of  a  rabbit  cause 
death  in  less  than  twenty-four  hours.  On  autopsy  intense 
hyperaBmia  of  the  peritoneum,  ecchymotic  'spots  of  the  intes- 
tines, swelling  of  Peyer's  patches,  and  enlargement  of  the 
spleen  are  found.  Subcutaneous  inoculations  are  followed  by 
abscesses  formed  at  the  point  of  inoculation  and  by  internal 
conditions  similar  to  those  produced  by  intravascular  injec- 
tions. Injected  into  the  pleural  cavity  it  gives  rise  in  twenty- 
four  hours  to  a  purulent  pleurisy  accompanied  by  a  large 
effusion  in  the  cavity  and  the  formation  of  false  membrane. 


168  ASIATIC  CHOLERA. 

QUESTIONS. 

When  and  by  whom  was  the  Bacillus  coli  communis  discovered  ? 

Where  is  it  found  in  the  body  in  health  ?     In  pathological  conditions? 

What  pathological  conditions  are  found  to  be  due  to  the  presence  of  this 
microorganism? 

Where  is  it  found  outside  the  human  body  ? 

Describe  the  Bacillus  coll  communis. 

How  do  its  flagella  compare  with  those  of  the  typhoid  bacillus  ? 

How  is  it  stained  ? 

How  does  it  behave  in  reference  to  oxygen  ? 

What  is  peculiar  about  its  motility  ? 

How  does  it  grow  on  artificial  media  and  at  what  temperature? 

What  is  its  thermal  death -point? 

What  is  the  effect  of  cold  ? 

How  does  it  grow  in  bouillon ?  On  gelatin?  On lactose-litmus-agar ?  On 
potato?  In  milk?  In  Donovan's  peptone  solution  ? 

What  is  the  effect  of  intraperitoneal  and  intravascular  inoculations  in 
animals  ? 

What  lesions  are  found  at  the  autopsy? 

What  lesions  are  produced  by  subcutaneous  inoculation  ?  By  intrapleural 
inoculation  ? 


CHAPTEK  XVIII. 
ASIATIC  CHOLERA. 

Spirillum  Gholerae  Asiatic ae  (Comma  Bacillus). 

History. — In  the  Cholera  Congress  at  Berlin,,  1884,  Koch 
made  the  announcement  that  he  had  been  able  to  isolate  from 
the  intestinal  dejecta  of  cholera  patients  a  microorganism 
which  he  believed  to  be  the  cause  of  the  disease.  His  experi- 
ments were  carried  out  in  a  number  of  cholera-infested 
places  and  on  a  large  number  of  patients.  His  conclusions, 
though  very  much  questioned  at  the  time,  are  to-day  accepted 
by  all,  and  his  Spirillum  cholerce  Asiaticce,  more  commonly 
known  as  the  comma  bacillus,  is  recognized  as  the  etiological 
factor  in  Asiatic  cholera. 

Morphology. — This  microorganism  belongs  to  the  class  of 
spirilla  called  by  some  authorities  vibrios. 

It  is  found  in  the  secretions  of  cholera  patients  and  in  cult- 


SPIRILLUM  CHOLERA  ASIATICS.  169 

ures  as  a  short  curved  rod,  from  0.8  to  2  mikrons  in  length, 
by  0.3  to  0.4  niikron  in  breadth.  Sometimes  two  of  these 
rods  are  united  together  by  either  end,  with  the  convex  sur- 
face looking  different  ways,  appearing  then  as  the  Roman 
letter  S ;  at  other  times  a  number  of  the  rods  are  united 
together  forming  a  long  spirillum.  These  latter  forms  are 
especially  seen  in  older  cultures.  In  young  cultures  the  rods 
are  generally  single  or  lying  together  and  parallel  to  each 
other.  This  peculiar  mode  of  grouping  serves  in  the  recogni- 
tion of  this  bacterium. 

The  Spirillum  cholerce  Asiatics  stains  with  all   the  anilin 
dyes,  but   rather  poorly.     It  seems  to  have  a  more  active 

FIG.  66. 


Spirillum  of  Asiatic  cholera.  Impression  Involution-forms  of  the  spirillum 
cover-slip  from  a  colony  thirty-four  hours  of  Asiatic  cholera,  as  seen  in  old 
old.  (Abbott.)  cultures.  (Abbott.) 

affinity  for  the  fuchsin  dye.  It  does  not  stain  by  the  Gram 
method.  Young  cultures  take  the  stain  much  more  readily 
than  older  cultures,  and  in  these  what  is  known  as  involution- 
forms — long,  thready  filaments  of  different  thickness — are  often 
found.  The  spirillum  contains  no  spores,  but  has  a  single 
flagellum  at  each  end  (Figs.  66  and  67). 

Biologic  Characters. — The  comma  bacillus  is  strictly  aerobic, 
and  though  it  grows  in  an  atmosphere  in  which  the  oxygen 
is  diminished,  it  can  not  grow  in  the  absence  of  this  gas. 
This  fact  is  the  cause  of  its  surface  growth  in  fluid  media. 

It  is  an  artificially  motile  spirillum,  especially  when  lately 
obtained  from  cholera  cases  or  in  young  cultures.  It  grows 


170 


ASIATIC  CHOLERA. 


in  all  artificial  media,  provided  these  are  neutral  or  slightly 
alkaline. 

Its  growth  on  gelatin  plates  and  stab-cultures  is  quite 
characteristic.  At  the  end  of  a  few  hours  on  gelatin  plates 
the  colony  appears  as  a  light  whitish  point,  which  grows 
very  rapidly,  liquefying  slightly  the  gelatin  around  it.  This 

FIG.  68. 


«  b  c  d 

Stab-culture  of  the  spirillum  of  Asiatic  cholera  in  gelatin,  at  18°  to  20°  C. :  a,  after 
twenty-four  hours ;  6,  after  forty -eight  hours ;  c,  after  seventy-two  hours  •  d  '  after 
ninety-six  hours.  (Abbott.) 

liquefaction  of  the  gelatin  seems  to  be  accompanied  by 
evaporation  of  the  liquid,  so  that  the  colony  sinks  into  the 
depth  of  the  space  left  in  the  gelatin  by  the  liquefaction,  and 
the  whole  surface  of  the  plate  seems  to  be  punched  out.  In 
gelatin  stab-cultures  the  surface  growth  shows  liquefaction  of 
the  gelatin  around  the  colony,  and  this  liquefaction  gradually 


SPIRILLUM  CHOLERA  ASIATICS.  171 

enlarges  and  extends  along  the  track  of  the  inoculating 
needle,  being  broader  at  the  surface  and  forming  a  short 
funnel,  which  from  the  evaporation  of  the  liquefied  gelatin  at 
the  top  gives  it  a  characteristic  appearance  (Fig.  68). 

Its  growth  on  agar  resembles  very  much  the  growth  on 
gelatin,  but  the  medium  is  not  liquefied.  Milk  is  coagulated 
by  the  formation  of  acids  in  the  medium.  In  peptone- 
bouillon  the  medium  is  clouded,  and  a  pellicle  forms  on  the 
surface. 

Vitality. — Its  growth  is  very  rapid,  and  advances  best  at  a 
temperature  between  35°  and  37°  C.,  but  continues  at  a  tem- 
perature as  low  as  17°  C.  Its  growth  is  stopped  at  a  tem- 
perature of  16°  C.,  and  the  bacterium  is  destroyed  in  five 
minutes  by  an  exposure  to  65°  C.  Freezing  does  not  destroy 
it.  Dryness  destroys  it  very  rapidly,  but  in  the  moist  state 
it  may  be  kept  frequently  for  several  days  and  sometimes  for 
several  months. 

Rapidity  of  Growth. — When  associated  with  other  bacteria 
in  cultures  it  grows  at  first  much  more  readily  than  any  of 
the  known  bacteria,  having  a  tendency  to  form  a  surface 
growth.  At  the  end  of  eighteen  to  twenty  hours,  however, 
it  is  outstripped  in  its  growth  by  the  other  bacteria,  and  in 
twenty-four  to  forty-eight  hours  its  growth  ceases  altogether, 
and  in  a  few  days  scarcely  any  spirillum  may  be  found  in 
the  cultures.  This  is  not  due  to  the  fact  that  it  is  destroyed 
by  its  association  with  the  other  bacteria,  but  more  because 
the  pabulum  necessary  for  its  growth  is  consumed.  The 
rapidity  of  the  growth  of  this  bacterium  and  the  fact  of 
its  growing  on  the  surface  of  liquids  are  a  great  help  in  its 
isolation  from  cholera  dejecta,  which  when  diluted  with  a 
large  amount  of  peptone-bouillon  shows  in  a  few  hours  a 
peculiar  surface  growth,  which  consists  almost  of  a  pure 
culture  of  cholera  spirilla. 

Pathogenesis. — None  of  the  domestic  animals  contracts  the 
disease  naturally.  But  their  immunity  seems  to  be  due 
to  the  fact  that  the  bacteria  that  they  ingest  at  the  time 
that  they  are  exposed  are  destroyed  by  the  acidity  of  the 
gastric  juice. 


172  ASIATIC  CHOLERA. 

Artificial  Susceptibility. — A  number  of  ingenious  devices 
have  been  resorted  to  to  render  animals  susceptible  to  inocula- 
tion of  the  comma  bacillus. 

The  method  of  Koch  is  ingenious  and  very  successful.  It 
consists  in  neutralizing  the  acidity  of  the  gastric  juice  in  a 
guinea-pig  by  the  inoculation  of  10  c.c.  of  a  5  per  cent, 
solution  of  carbonate  of  sodium.  This  is  introduced  into  the 
stomach  by  means  of  a  soft  catheter.  A  few  minutes  after- 
ward 10  c.c.  of  young  bouillon  cultures  of  the  cholera  spiril- 
lum are  introduced  also  into  the  stomach  through  the  same 
catheter,  and  immediately  an  intraperitoneal  injection  of  1  c.c. 
of  laudanum  is  made  into  the  animal,  for  the  purpose  of  retard- 
ing peristaltic  action.  The  animal  for  an  hour  or  so  remains 
in  a  stupefied  condition  from  the  action  of  the  opiate,  but  it 
soon  revives.  It  shows,  however,  a  complete  loss  of  appetite, 
and  at  the  end  of  twenty-four  hours  begins  to  show  signs  of 
paralysis  of  the  hind  extremities,  with  coldness  of  the  sur- 
face. This  paralysis  gradually  increases  until  in  forty-eight 
hours  the  animal  dies,  showing  pathologically  some  lesions 
resembling  those  found  in  man  in  cases  of  cholera — i.  e.,  a 
large  amount  of  white  serous  exudate  in  the  intestinal  canal, 
with  intense  congestion  of  the  intestines.  Pure  colonies  of 
the  spirillum  may  be  obtained  from  these  secretions. 

Intraperitoneal  injections  in  animals  are  followed  by  death 
in  two  or  three  hours.  The  symptoms  are  those  of  a  rapid 
and  intense  peritonitis. 

Immunity. — When  the  injections  into  animals  are  made  in 
quantities  too  small  to  produce  death  the  animal  is  protected 
for  a  time  from  subsequent  fatal  doses,  and  its  serum  has 
been  found  useful  to  protect  animals  of  the  same  species 
against  inoculations  with  fatal  doses  of  the  bacteria. 

The  blood-serum  of  these  immunized  animals,  as  well  as 
that  of  cholera  patients,  has  been  found  in  a  dilution  of  1  to 
50  to  possess  the  power  of  agglutination  when  mixed  with 
young  bouillon  cultures  of  the  bacteria.  This  may  be  used 
as  a  diagnostic  test  of  the  disease. 

The  organism  is  seldom  or  never  found  in  the  general  cir- 
culation nor  in  the  internal  organs  of  cholera  cases. 


QUESTIONS.  173 

Diagnosis. — For  this  purpose  the  rate  of  the  growth  on 
gelatin  plates  and  the  rapidity  of  the  indol-formation  in 
Dunham's  solution  are  made  use  of.  The  experiments  are 
carried  on  as  follows  : 

The  small  flocculent  masses  found  in  the  discharges  of 
choleraic  patients  are  taken  and  mixed  with  a  large  quantity 
of  diluted  peptone-bouillon,  or  preferably  with  Dunham's 
solution  of  peptone,  and  put  into  the  incubator  for  three  or 
four  hours.  At  the  end  of  that  time  a  few  drops  from  the 
surface  of  the  liquid  are  taken  and  inoculated  on  gelatin 
plates,  when  characteristic  colonies  are  developed  in  a  few 
hours.  Cover-glass  preparations  are  also  made,  and  if  rods 
with  a  morphological  appearance  of  cholera  bacteria  are  found, 
agar  plates  are  also  made  in  this  way.  Melted  agar  is  poured 
into  Petri  dishes,  and  these  put  into  the  incubator  for  a  few 
hours  in  order  to  allow  the  evaporation  water  to  collect  on  the 
surface  of  the  agar ;  this  water  is  poured  off  and  the  dishes 
inoculated  by  streaking  the  surface  with  the  suspected  material. 
In  a  very  short  time  characteristic  colonies  develop  along  the 
line  of  the  streak. 

The  cholera  bacteria  are  of  very  rapid  growth,  but  possess 
little  or  no  resisting  power,  being  destroyed  by  the  physical 
measures  just  mentioned,  and  also  in  a  very  short  time  by 
the  use  of  weak  disinfectants. 

Vaccinations  against  cholera  have  been  performed  on  an  ex- 
tensive scale  in  cholera-infected  countries.  Haffkine's  method 
of  injecting  attenuated  or  small  doses  of  virulent  cultures  of 
the  cholera  spirillum  as  a  means  of  protection  against  an 
attack  of  cholera  seems  to  have  rendered  considerable  service 
in  protecting  persons  exposed  to  the  disease  ;  and  experiments 
made  by  Ferran,  in  Spain,  with  attenuated  cultures  seem  to 
have  given  encouraging  results  at  the  time  of  the  cholera 
visitation. 

QUESTIONS. 

When  and  by  whom  was  the  Spirillum  choleras  Asiaticse  (comma  bacillus) 
discovered  ? 

Describe  the  spirillum. 

What  is  the  peculiar  arrangement  of  the  bacteria  in  cultures  and  secre- 
tions? 


174  INFLUENZA. 

How  does  the  comma  bacillus  stain  ? 

Does  it  contain  spores? 

Has  it  flagella  ? 

How  does  it  behave  in  the  presence  of  oxygen ? 

Is  it  motile? 

What  condition  of  the  media  is  necessary  for  its  growth  ? 

How  does  it  grow  on  gelatin  plates ?  In  stab- cultures?  On  agar?  In  pep- 
tone-bouillon ? 

At  what  temperature  does  it  grow? 

What  is  its  thermal  death-point  ? 

What  is  the  effect  of  cold  ?    Of  dryuess  ? 

How  long  may  it  be  kept  in  a  moist  state  ? 

How  does  it  grow  when  associated  with  other  bacteria? 

What  peculiarities  of  its  growth  are  made  use  of  in  those  cases  to  isolate  it? 

What  is  the  cause  of  the  natural  immunity  of  domestic  animals  to  cholera  ? 

How  has  Koch  succeeded  in  inoculating  the  lower  animals  through  the 
stomach  ? 

What  effect  has  inoculation  of  animals  with  cultures  of  the  comma  bacillus  ? 

What  is  the  effect  of  intraperitoneal  inoculation  ? 

How  are  animals  made  immune  against  cholera  inoculation  ? 

What  is  the  effect  of  the  blood-serum  of  immunized  animals  on  other 
animals? 

Where  are  the  organisms  found  in  cholera  patients  or  at  the  autopsy  in  a 
cholera  case  ? 

How  is  the  cholera  bacillus  isolated  from  cholera  dejecta? 

How  much  resisting  power  has  the  comma  bacillus? 

What  is  Haffkine's  method  of  protection  against  cholera? 


CHAPTEK  XIX. 

INFLUENZA. 

Bacillus  of  Influenza. 

History. — In  1892  Pfeiffer  and  Cannon  independently  iso- 
lated from  the  bronchial  and  nasal  secretions  of  cases  of 
influenza,  and  from  the  blood  in  some  cases,  a  small  micro- 
organism which  they  believed,  with  apparent  correctness,  to 
be  the  cause  of  the  disease. 

Morphology. — The  bacillus  so  isolated  may  be  described  as 
follows  :  a  small,  thick  rod,  occurring  singly  or  in  pairs, 
stained  with  difficulty  by  the  ordinary  anilin  dyes,  but  fairly 
well  with  a  diluted  Ziehl  solution  or  Loeffler's  methylene- 
blue ;  not  stained  by  Gram's  method.  The  body  of  the  rod 
stains  less  well  than  the  ends.  It  has  no  flagella  and  contains 
no  spores.  (Plate  IV.) 


PLATE  IV. 


.*\ 

*wv 

V 


Bacillus  of  Influenza  in  Sputum.    (Abbott.) 


QUESTIONS.  175 

Biologic  Characters. — The  bacillus  of  influenza  is  strictly 
aerobic,  not  growing  at  all  without  oxygen.  It  is  non-motile, 
and  grows  at  a  temperature  between  26°  and  43°  C. 

It  grows  but  rather  poorly  in  all  media  that  may  be  sub- 
mitted to  this  temperature,  unless  the  surface  of  the  media 
be  smeared  over  with  fresh  sterilized  blood,  when  the  growth 
is  quite  luxuriant.  On  glycerin-agar  or  in  blood-serum  tubes 
on  which  fresh  rabbit's  blood  has  been  smeared,  it  grows  as 
transparent  watery  colonies,  resembling  very  much  dew- 
drops.  The  colonies  have  no  tendency  to  coalesce.  In 
bouillon  to  which  a  little  fresh  blood  has  been  added  it  grows 
luxuriantly.  It  does  not  cause  clouding  of  the  medium,  but 
its  colonies  are  seen  as  little  flakes  adhering  to  the  sides  of 
the  tube  and  forming  a  deposit  at  the  bottom. 

Vitality. — The  bacillus  of  influenza  is  destroyed  in  two  or 
three  hours  by  drying.  It  has  very  little  resisting  power, 
and  in  water  lives  scarcely  twenty-four  hours.  In  pneu- 
monia occurring  during  the  course  of  this  disease  the  bacilli 
are  often  found  in  the  body  of  the  leucocytes. 

Pathogenesis. — Outside  of  the  human  race  none  of  the 
lower  animals  seems  to  be  susceptible  to  the  disease,  excepting 
perhaps  the  monkey,  and  by  inoculation  it  is  difficult  to 
produce  any  symptom  in  laboratory  animals.  In  man,  how- 
ever, the  bacillus  is  constantly  found  in  the  bronchial  and 
nasal  secretions,  also  in  the  pneumonic  patches  so  often  found 
in  the  course  of  this  disease.  At  autopsies  it  has  been  found 
also  in  the  spleen  and  occasionally  in  the  blood.  Some  per- 
sons have  a  natural  power  of  retaining  live  bacilli  in  the 
lungs  for  a  considerable  length  of  time;  especially  is  this  the 
case  with  tuberculous  patients,  in  whose  sputum  is  very  often 
found  the  Bacillus  influenzas.  By  inoculating  animals  in  the 
brain,  the  nervous  phenomena  of  this  disease  have  been 
easily  reproduced. 

QUESTIONS. 

By  whom  and  when  was  the  bacillus  of  influenza  discovered  ? 

Describe  this  bacillus  and  its  staining  peculiarities? 

Does  it  possess  flagella  ?     Spores  ? 

What  are  the  principal  biologic  characters  of  the  bacillus  of  influenza  ? 


176  BUBONIC  PLAGUE. 

At  what  temperature  does  it  grow  ? 
In  what  artificial  media  does  it  grow  ? 
What  must  be  added  to  this  media  to  facilitate  its  growth  ? 
What  is  the  appearance  of  the  colonies,  in  glycerin  ?  On  agar  ?   In  blood- 
serum  ? 

How  does  it  grow  in  bouillon  ? 

What  is  the  resisting  power  of  this  bacillus  ? 

What  animals  are  susceptible  ? 

Where  is  the  bacillus  found  in  animals? 

What  is  peculiar  about  the  retention  of  this  bacillus  by  some  persons? 

How  may  this  explain  the  spread  of  the  disease  ? 


CHAPTER    XX. 
BUBONIC   PLAGUE. 

Bacillus  Pestis. 

History. — Under  various  names  and  from  the  remotest 
times  epidemics  of  bubonic  plague  have  appeared  in  the  old 
world,  causing  an  immense  fatality. 

Yersin  and  Kitasato,  in  1894,  working  independently,  have 
both  discovered  the  pathogenic  germ  of  this  disease  in  the 
suppurating  buboes,  blood,  internal  organs,  and  excretions  of 
persons  affected,  and  called  it  the  Bacillus  pestis. 

Morphology. — This  bacillus  is  a  short,  oval,  thick  rod, 
occurring  singly  or  in  pairs,  or  sometimes  by  the  union  of  a 
number  forming  long  filaments  or  threads.  Staining  with 
all  the  anilin  dyes,  but  not  by  Gram's  method.  In  stained 
preparations  the  centre  of  the  bacilli  cell  stains  less  well 
than  the  ends  of  the  rod,  giving  it  quite  a  characteristic 
appearance.  (Plate  V.) 

This  bacillus  has  no  flagella. 

Biologic  Characters. — The  Bacillus  pestis  is  a  non-motile 
aerobic.  It  grows  at  all  temperatures,  but  best  between  36° 
and  39°  C.  It  is  killed  by  a  temperature  of  80°  C.  after  an 
exposure  of  a  half-hour,  and  in  five  minutes  by  an  exposure 
to  100°  C.  in  the  steam  sterilizer.  It  grows  in  all  the  arti- 


PLATE  V. 


Bacillus  of  Bubonic  Plague      (Abbott.) 

A.  In  pus  from  suppurating  bubo.     B.  The  bacillus  very  much  enlarged 
to  show  peculiar  polar  staining. 


BACILLUS  PESTIS.  177 

ficial  media.  On  gelatin,  after  twenty-four  or  thirty-six 
hours  the  colonies  appear  as  small,  sharply  defined,  round, 
white  masses  which  do  not  liquefy  the  medium.  Its  growth 
in  agar  in  the  incubator  is  a  little  more  rapid  than  in  gelatin. 
It  <loes  not  cloud  bouillon.  Cultures  in  this  medium  show  a 
number  of  flocculi  in  the  tube  and  a  deposit  at  the  bottom. 
It  does  not  cause  fermentation,  and  it  gives  no  indol  reaction. 
It  coagulates  milk. 

Vitality. — The  Bacillus  pestis  is  very  susceptible  to  the 
action  of  disinfectants,  1  per  cent,  carbolic  acid  being  suf- 
ficient to  kill  it  in  one  hour. 

Pathogenesis. — Man,  mice,  rats,  guinea-pigs,  rabbits,  cats, 
hogs,  horses,  chickens,  and  sparrows  are  very  susceptible  to 
the  disease.  Pigeons,  dogs,  amphibia,  and  bovines  appear  to 
enjoy  immunity.  During  an  epidemic  of  bubonic  plague  sus- 
ceptible animah  seem  to  contract  the  disease  naturally. 

For  experimentation  subcutaneous  inoculation  with  liquid 
cultures  of  the  bacillus  is  generally  resorted  to.  The  changes 
produced  are  :  Swelling  of  cedematous  character  at  the  point 
of  inoculation,  and  involvement  of  the  lymphatic  glands; 
death  resulting  in  from  twenty- four  to  forty-eight  hours.  At 
the  autopsy  the  local  swelling  is  found  to  be  due  to  an  cedern- 
atous  condition  of  the  part,  the  bloody  fluid  containing  a 
large  number  of  bacilli.  The  neighboring  lymphatic  glands 
are  also  greatly  inflamed,  and  some  of  them  are  found  sup- 
purating. In  their  substance  the  pest  bacilli  are  also  found 
in  great  number.  There  also  occurs  a  purulent  exudate  in 
the  peritoneal  and  pleural  cavities.  The  internal  organs,  liver, 
lungs,  adrenal  bodies,  and  spleen  are  very  much  affected. 

Three  forms  of  the  disease  are  recognized  in  man  :  the 
bubonic  or  ganglionic,  the  septicsemic,  and  the  pneumonic  form, 
the  most  frequent  of  these  being  the  bubonic,  and  the  most 
fatal  the  pneumonic. 

Infection  generally  takes  place  through  an  abrasion  of  the 
skin,  but  the  disease  may  be  caused  by  inhalation  of  the  pest 
bacilli. 

The  usual  form  of  the  disease  presents  the  following  symp- 
toms :  a  sudden  rise  of  temperature  accompanied  by  great 
12— M.  B. 


178  BUBONIC  PLAGUE. 

prostration  and  delirium,  and  the  occurrence  of  lymphatic 
swellings  (buboes)  affecting  chiefly  the  glands  corresponding 
to  the  inoculated  portion.  These  become  very  much  enlarged, 
and  have  a  tendency  to  soften  and  suppurate.  In  severe 
cases  death  occurs  in  forty-eight  hours ;  in  others,  the  dura- 
tion of  the  disease  is  somewhat  longer.  The  prognosis  is 
more  favorable,  the  longer  the  duration  of  the  disease.  Char- 
acteristic bacilli  are  found  in  the  lymphatic  glands,  and  also 
occasionally  in  the  blood. 

Immunity. — Persons  who  have  recovered  from  an  attack  of 
bubonic  plague  or  animals  that  have  survived  inoculations 
are  found  to  be  immune  for  a  certain  period  of  time.  This 
immunity  is  due  to  a  substance  developed  in  the  serum  of 
those  animals,  which  may  also  when  inoculated  into  suscepti- 
ble animals  protect  them  from  infection  with  bubonic  plague. 
Artificial  immunity  may  also  be  conferred  by  injecting  cult- 
ures of  the  dead  bacilli. 

Agglutination. — The  serum  of  immune  animals  possesses 
also  an  agglutinating  action  when  mixed  with  bouillon  cult- 
ures of  the  Bacillus  pestis,  very  much  the  same  as  the  agglu- 
tinative action  of  typhoid  or  cholera  serum. 

Serotherapeutics. — Yersin  claims  to  have  developed  a  serum 
in  the  horse  which  is  not  only  protective,  but  also  curative, 
when  injected  into  the  human  being.  His  experiments, 
carried  on  in  a  number  of  cases,  seem  to  indicate  this  serum 
to  have  some  decided  beneficial  effect  when  administered  early 
in  the  disease,  the  proportion  of  deaths  in  cases  so  treated 
being  scarcely  8  per  cent.,  whereas  the  mortality  in  non- 
inoculated  cases  is  as  high  as  80  per  cent.  To  obtain  the 
blood-serum  from  horses,  he  immunizes  them  with  the  dead 
bouillon  cultures  of  the  Bacillus  pestis. 

Haffkine  has  practised  on  an  extensive  scale  protective  in- 
oculations against  bubonic  plague  by  injections  of  dead  cult- 
ures. This  immunity  seems  to  last  for  several  weeks. 

Recently,  observations  have  demonstrated  that  Yersin 
serum  has  absolutely  no  protective  or  curative  properties. 
Haffkine's  protective  inoculations  are  still  held  in  favor, 
however. 


RELAPSING  FEVER.  179 

QUESTIONS. 

By  whom  and  when  was  the  Bacillus pestis  discovered? 

Describe  this  bacillus.  Its  mode  of  staining.  Its  principal  biologic  char- 
acters. 

Wliat  temperature  suits  its  growth  best?  What  is  the  effect  of  high  tem- 
perature? 

How  does  it  grow  on  gelatin  ?     On  agar  ?     On  bouillon  ? 

What  is  its  behavior  with  regard  to  fermentation  and  to  indol  pro- 
duction? 

How  does  it  affect  milk? 

How  is  it  affected  by  disinfectants? 

What  animals  are  susceptible  to  this  disease? 

How  are  inoculations  performed  ? 

Describe  the  symptoms  and  lesions  caused  by  inoculation. 

What  three  forms  of  bubonic  plague  are  recognized  in  man?  Which  is 
the  most  frequent?  Which  the  most  fatal? 

How  does  infection  take  place? 

What  are  the  symptoms  of  the  disease,  and  what  the  lesions  in  man? 

How  is  immunity  conferred  in  this  disease? 

Does  the  serum  in  cases  of  plague  contain  agglutinating  power? 

How  does  Yersin  manufacture  his  protective  serum?  What  does  he  claim 
for  it  ? 

How  does  Haff kine  practise  his  protective  inoculations  ? 


CHAPTER  XXI. 
RELAPSING  FEVER. 

Spirillum  Obermeieri. 

History. — As  early  as  1873  Obermeier  discovered  in  the 
blood  of  patients  suffering  from  relapsing  fever  a  long,  spi- 
rillum-like microorganism,  measuring  from  20  to  30  mikrons, 
having  the  power  of  active  motion.  His  observations  have 
since  been  confirmed  by  a  number  of  other  investigators. 

This  spirillum,  which  has  not  been  cultivated  artificially,  is 
found  in  the  blood  and  spleen,  but  never  in  the  secretions  of 
patients  affected  with  relapsing  fever. 

Morphology  and  Biology. — It  stains  readily  by  all  the  anilin 
dyes,  but  does  not  stain  by  Gram's  method.  It  is  actively 
motile  and  contains  no  spores. 

In  the  blood  it  is  found  in  two  forms:  (1)  during  the  pyrexia 


180       DYSENTERY,  HOG  AND  CHICKEN  CHOLERA. 

as  long  twisted  filaments ;  (2)  after  the  crisis  of  the  fever  is 
reached  it  is  seen  in  the  leucocytes  as  short  degenerated 
curved  rods. 

Pathogenesis. — This  spirillum  is  not  pathogenic  to  the  lower 
animals,  with  the  exception  of  the  monkey. 

Blood  taken  from  patients  during  the  paroxysm  when  in- 
oculated in  other  individuals  may  give  rise  to  relapsing  fever. 

One  attack  of  the  disease  does  not  seem  to  confer  immunity 
from  future  attacks,  but  rather  renders  the  subject  more 
susceptible. 

QUESTIONS. 

What  microorganism  is  the  cause  of  relapsing  fever? 
By  whom  and  when  was  it  discovered  ? 
Describe  it. 
How  does  it  stain  ? 
May  it  be  cultivated  artificially? 
Where  is  it  found  in  cases  of  relapsing  fever? 
Is  it  motile  ? 
Does  it  contain  spores? 

In  what  two  forms  is  it  found  in  the  blood  ? 
For  what  animals  is  it  pathogenic  ? 

May  blood  of  relapsing  fever  give  rise  to  other  cases  of  the  disease  by 
inoculation  ? 

Does  one  attack  of  relapsing  fever  confer  immunity  upon  the  subject  ? 


CHAPTER   XXII. 

DYSENTERY,  HOG  CHOLERA,  AND  CHICKEN  CHOLERA. 
DYSENTERY. 

Bacillus  Dysentericse. 

History. — This  bacillus  was  first  found  in  the  intestinal  con- 
tents and  in  the  visceral  walls  and  mesenteric  glands  in  cases 
of  acute  epidemics  of  dysentery  by  Shiga,  in  Japan  in  1898, 
and  this  observation  was  confirmed  afterward  by  Flexner  in  a 
study  of  dysentery  of  the  Philippine  Islands.  It  seems  to 
belong  to  the  typho-colon  group. 


DYSENTERY.  181 

Morphology. — The  Bacillus  dysentericce  is  of  medium  size, 
with  round  ends,  containing  no  spores,  and  having  flagella.  It 
stains  by  the  ordinary  anilin  dyes,  but  not  by  Gram's  method. 

Biologic  Characters. — It  is  aerobic,  but  may  be  grown  with- 
out oxygen.  It  grows  at  the  ordinary  room  temperature,  but 
best  at  the  temperature  of  the  human  body,  and  does  not 
liquefy  gelatin. 

Its  growth  on  agar  is  not  characteristic,  slightly  resembling 
the  typhoid  growth,  and  on  gelatin  the  growth  is  pearl  col- 
ored somewhat  like  the  typhoid,  but  later  becomes  moist. 
On  potato  it  sometimes  has  also  an  invisible  growth ;  at  other 
times  its  growth  is  rather  voluminous  and  grayish  brown  in 
color.  It  clouds  bouillon  without  forming  a  pellicle  on  the 
surface.  It  causes  no  fermentation,  though  it  causes  a  slight 
increase  of  acidity  in  glucose-bouillon.  It  does  not  liquefy 
blood-serum.  Litmus  milk  at  the  end  of  three  days  becomes 
of  a  pale  lilac  color,  but  the  milk  is  not  coagulated.  In  six 
or  seven  days  the  medium  becomes  dark  blue.  It  produces 
no  indol. 

Agglutination. — The  serum  of  affected  animals  has  an 
agglutinating  power  on  young  cultures  of  the  bacillus. 

It  is  pathogenic  for  laboratory  animals.  When  injected 
intraperitoneally  into  animals  it  produces  a  purulent  peri- 
tonitis, with  involvement  of  the  mesenteric  glands  and  swollen 
spleen,  and  the  liver  is  covered  with  an  exudate.  The  intes- 
tinal glands  and  Peyer's  patches  show  signs  of  inflammation. 
The  bacillus  may  be  recovered  from  the  exudate,  and  also  in 
limited  quantity  from  the  organs.  Subcutaneous  injections 
show  swelling  and  o?dema  at  the  point  of  inoculation,  with 
involvement  of  the  lymphatic  glands,  and  are  also  followed 
by  effusion  in  the  serous  cavities. 

By  alkalinizing  the  secretions  of  the  stomach,  animals  have 
been  infected  by  feeding  with  the  bacillus,  and  in  those  ani- 
mals lesions  very  much  resembling  the  disease  in  man  have 
been  reproduced,  and  pure  cultures  of  the  bacillus  obtained 
from  the  secretions  of  the  intestines. 

That  the  poison  is  in  the  cell-body  of  the  Bacillus  dysen- 
tencce,  and  is  not  a  secretion  of  the  cells,  is  demonstrated  by 


182       DYSENTERY,  HOG  AND   CHICKEN  CHOLERA. 

the  fact  that  by  heating  cultures  to  a  temperature  above 
60°  C.,  which  kills  the  bacilli,  does  not  seem  to  have  any 
effect  on  the  activity  of  the  poison. 

Protective  inoculations  in  animals  have  been  performed  with 
positive  results,  and  the  serum  of  immunized  animals  has 
been  found  to  possess  the  power  of  agglutination,  and  to  be 
both  protective  and  curative. 

HOG  CHOLERA. 
Bacillus  Sui  Pestifer. 

History. — In  the  dejecta  of  hogs  affected  with  cholera 
Salmon  and  Smith  have  succeeded  in  isolating  a  bacillus 
which  they  found  to  be  the  specific  cause  of  this  disease. 

Morphology. — It  is  a  short,  thick  rod,  1.20  to  1.50  mikrons 
in  length,  and  0.6  to  0.7  mikron  in  breadth,  actively  motile, 
containing  flagella,  stains  by  all  the  anilin  dyes,  but  not  by 
Gram's  method. 

Biologic  Characters. — It  is  aerobic  and  grows  in  all  the  cult- 
ure-media. Its  growth  on  gelatin  is  visible  in  from  twenty- 
four  to  forty-eight  hours;  the  colonies  appear  irregularly 
round  and  the  gelatin  is  not  liquefied.  On  agar-agar  the 
colonies  are  translucent  and  rather  circumscribed.  Upon 
potato  the  colonies  are  yellow.  Bouillon  is  clouded  and  a 
thin  surface  growth  may  be  observed.  In  milk  it  does  not 
generate  acids  and  does  not  coagulate  it. 

It  produces  gas  copiously,  but  no  indol. 

Vitality. — It  withstands  drying  for  a  long  time.  Its  ther- 
mal death-point  is  54°  C. 

Pathogenesis. — It  is  intensely  pathogenic  for  every  labor- 
atory animal,  death  being  preceded  always  by  a  rise  of 
temperature,  and  postmortem  lesions  affecting  chiefly  the  liver 
and  kidneys  are  seen.  Sometimes  the  lesions  are  found  in 
the  intestines  and  Peyer's  glands  also.  The  bacillus  is  found 
in  all  the  organs.  Artificially  swine  are  inoculated  with  dif- 
ficulty. 

Immunity  in  animals  has  been  produced  by  Salmon  and 
Smith  by  injections  of  gradually  increasing  doses  of  cultures 


CHICKEN  CHOLERA.  183 

of  this  bacillus.  De  Schweinitz  has  isolated  from  cultures 
of  the  bacteria  pure  toxic  substances  with  which  he  has  been 
able  to  produce  immunity.  By  subcutaneous  inoculations 
with  these  toxins  in  cows  he  was  able  to  develop  in  their 
blood-serum  an  antitoxic  substance  capable  of  protecting  ani- 
mals from  the  disease.  The  serum  of  infected  animals  has  a 
remarkable  agglutinating  power;  with  a  dilution  of  1  to 
10,000,  agglutination  can  be  obtained  in  an  hour. 

CHICKEN  CHOLERA. 
Bacillus  Cholerse  Gallinarum. 

History. — This  bacillus  was  observed  by  Perroncito,  in 
1878,  and  described  by  Pasteur. 

The  cause  of  the  disease  known  in  fowls  as  chicken  cholera 
is  due  to  a  short,  broad  bacillus,  with  rounded  ends,  occurring 
singly  or  united  to  form  filaments. 

This  bacillus  stains  in  a  peculiar  way  with  the  anilin  dyes, 
its  two  poles  being  markedly  stained,  whereas  the  centre  of 
the  bacillus  is  scarcely  stained  at  all,  giving  it  very  much  the 
appearance  of  a  micrococcus,  which  it  wras  at  first  believed 
to  be  by  Pasteur.  It  does  not  stain  by  Gram's  method. 

Biology. — It  produces  no  spores  and  is  non-motile.  It  is 
easily  killed  by  heat  and  drying.  It  grows  on  all  ordinary 
culture-media.  On  gelatin  in  two  days  the  cultures  appear  to 
the  naked  eye  as  small  white  points ;  under  the  microscope 
the  colonies  are  granular  and  concentric.  It  does  not  liquefy 
gelatin.  In  stab-cultures  its  growth  on  this  media  resembles 
that  of  a  nail  with  a  flat  head,  the  head  of  the  nail  being 
closer  to  the  surface  of  the  medium  than  the  point. 

Its  growth  in  agar  and  bouillon  offers  nothing  character- 
istic. The  bacillus  is  strictly  aerobic. 

Pathogenesis. — Chickens,  geese,  pigeons,  sparrows,  mice, 
and  rabbits  are  susceptible  animals.  Guinea-pigs  are  im- 
mune. By  inoculation  the  disease  produced  in  susceptible 
animals  is  that  of  a  general  septicaemia,  the  bacillus  being 
found  in  the  blood  and  all  the  internal  organs. 

By  feeding  the  contaminated  material  to  animals  the  lesions 


184       DYSENTERY,  HOG  AND   CHICKEN  CHOLERA. 

are  limited  to  the  intestines,  having  the  appearance  of  true 
cholera. 

Protective  inoculations  have  been  performed  by  using  atten- 
uated cultures,  the  attenuation  being  arrived  at,  as  recom- 
mended by  Pasteur,  by  using  cultures  two  or  three  months 
old.  This  bacillus  has  been  used  extensively  in  Australia 
for  the  destruction  of  rabbits.  It  is  said  that  Avith  two 
gallons  of  a  bouillon  culture  as  many  as  2000  rabbits  may  be 
destroyed. 

This  bacillus  has  been  described  by  different  authors  under 
a  number  of  names  :  as  the  bacillus  of  rabbit  septicaemia,  by 
Koch  ;  the  bacillus  of  swine  plague,  by  Loeffler  ;  Bacilli  cuni- 
cucilidi,  by  Fluegge,  and  others. 

QUESTIONS. 
Dysentery. 

When  and  by  whom  was  the  Bacillus  dysentericse  discovered  and  described  ? 

What  is  its  morphology  ? 

How  does  it  stain  ? 

Give  its  principal  biologic  characters. 

How  does  it  grow  on  agar  ?    On  potato  ?    On  bouillon  ?    In  litmus  milk  ? 

Does  it  produce  indol? 

Does  its  serum  have  an  agglutinating  power  ? 

What  is  the  effect  of  an  intraperitoneal  injection  in  animals?  Of  a  sub- 
cutaneous injection  ? 

How  are  animals  affected  by  feeding  of  the  bacilli  ? 

Where  is  the  toxin  of  the  Bacillus  dysentericse  contained  ? 

May  animals  be  immunized  against  dysentery  by  inoculation  with  Bacil- 
lus dysentericse  ? 

What  claims  are  made  for  the  serum  of  inoculated  animals  ? 


Hog  Cholera. 

By  whom  was  the  bacillus  of  hog  cholera  discovered  ? 
Give  its  morphology.     Its  staining.     Its  principal  biologic  characters. 
How  does  it  grow  on   gelatin  and   agar  ?     On  potato  ?     In  bouillon  ?     In 
milk? 

Does  it  produce  gases? 

What  is  its  thermal  death -point? 

How  does  it  affect  laboratory  animals? 

Has  immunity  been  produced  by  injections  of  cultures  of  this  bacillus  ? 

Has  a  toxin  been  isolated  from  the  bacillus  of  hog  cholera  ? 

What  are  the  properties  of  the  serum  of  immunized  animals? 


PATHOGENIC  MICROORGANISMS.  185 

Chicken  Cholera. 

By  whom  was  the  Bacillus  cholerse  gallinamm  discovered,  and  by  whom 
described  ? 

Dt'scrilic  this  bacillus. 

HII\V  docs  it  stain  ? 

(Jive  its  principal  biologic  characters. 

What  is  the  appearance  of  the  cultures  on  gelatin  to  the  naked  eye?  How 
do  they  look  when  seen  under  the  microscope? 

What  animals  are  subject  to  infection?     Which  animals  are  immune? 

What  sort  of  infection  is  produced  in  animals  by  the  inoculation  of  this 
bacillus? 

What  is  effected  by  feeding  animals  with  material  contaminated  with  this 
bacillus? 

How  are  protective  inoculations  performed? 

To  what  extent  is  this  bacillus  pathogenic  to  rabbits? 

Under  what  different  names  has  this  bacillus  been  described  by  various 
authors  ? 


CHAPTER    XXIII. 

THE    PATHOGENIC    MICROORGANISMS    OTHER    THAN 
BACTERIA. 

ACTINOMYCOSIS,  MALARIA,  AND  AMOEBIC  COLITIS. 

Streptothrix. 

THE  streptothrix  group,  which  has  not  as  yet  been  clearly 
defined,  presents  a  number  of  varieties  which  have  been 
found  pathogenic.  This  group  of  microorganisms  resembles 
the  bacteria,  yet  differ  from  them  in  a  number  of  important 
respects,  and  are  associated,  on  the  other  hand,  with  the 
moulds.  They  resemble  the  moulds  in  so  far  that  they  develop 
from  spore-like  bodies  into  dichotomously  branching  threads, 
which  grow  into  colonies  having  more  or  less  the  appearance 
of  true  mycelia.  Under  favorable  conditions  some  of  the 
threads  become  fruit  hyphse  and  break  up  into  a  number  of 
spore-like  bodies.  These  sporiform  bodies  differ  from  bac- 
terial spores  in  the  fact  that  they  are  stained  by  the  ordinary 
method  of  staining.  They  resemble  the  bacteria  in  the  fact 


186  PATHOGENIC  MICROORGANISMS. 

that  they  occur  as  threads  which  may  under  careful  cultiva- 
tion become  divided  into  short  segments.  They  do  not  have 
a  double  wall  like  the  moulds,  and  are  not  filled  with  fluid 
containing  granules,  and  the  segments  of  the  filaments  have 
no  distinct  partition  separating  one  from  the  other. 

The  most  thoroughly  studied  streptothrices  are  :  the  Strep- 
tothrix  actinomyces,  or  ray  fungus,  the  Streptothrix  madurce, 
and  the  Streptothrix  Eppingeri,  all  of  which  have  been  found 
associated  with  important  pathological  lesions  and  are  be- 
lieved to  be  the  cause  of  special  diseases.  The  bacteria  of 
tuberculosis  and  diphtheria  are  believed  by  some  to  belong  to 
the  class  of  streptothrices,  because  at  times  they  show  a  tendency 
to  form  branched  segments.  This  view,  however,  is  not  generally 
accepted.  It  is  interesting  to  note  that  the  lesions  caused  by  the 
streptothrix  have  very  much  the  appearance  of  tuberculous 
lesions,  being  almost  indistinguishable  from  tuberculosis  except 
for  the  absence  of  the  Bacillus  tuberculosis. 

In  diseases  attributable  to  these  fungi,  microscopical  exam- 
inations of  the  tissues  reveal  the  streptothrices  in  the  tissues, 
and  these  have  been  cultivated  artificially,  and  by  inocula- 
tions into  animals  have  reproduced  lesions  identical  with  those 
of  the  original  disease. 

The  most  important  of  the  diseases  caused  by  one  of  the 
streptothrices,  and  the  only  one  which  will  be  studied  in  this 
volume,  is  : 

ACTINOMYCOSIS. 
Streptothrix  Actinomyces  (Ray  Fungus). 

History. — It  was  described  first  by  Bollinger,  in  1877,  and 
found  in  the  disease  of  cattle  known  as  big-jaw  or  wooden- 
tongue,  and  contained  in  the  tissues  and  exudates.  In  man 
the  disease  first  described  by  Israel,  in  1885,  seems  to  be  iden- 
tical with  this  cattle  disease. 

Morphology. — In  pus  from  the  affected  parts  are  small  yel- 
lowish granular  masses  from  2  to  5  millimeters  in  diameter. 
Under  the  microscope  these  granules  are  seen  to  consist  of  a 
number  of  threads  which  radiate  from  a  centre  to  a  bulbous, 


ACTINOMYCOSIS.  187 

club-like  termination,  the  whole  mass  having  very  much  the 
appearance  of  a  rosette.  Sometimes  the  free  ends  of  the 
thread  are  only  slightly  or  not  at  all  swollen  (Fig.  69).  The 
threads  which  compose  the  centre  of  the  mass  are  from  0.3 
to  0.5  mikron  in  width.  The  clubs  are  from  0.6  to  0.8 
mikron  in  width.  These  mycelia  stain  by  all  the  ordinary 
anilin  colors,  and  also  by  Gram's  method,  though  by  these 
methods  their  fine  structure  is  not  always  brought  out.  The 
best  stain  for  the  fungus  is  Mallory's  stain,  which  is  as  fol- 
lows : 

Stain  the  secretions  on  the  slide  with  gentian-violet,  dehy- 
drate, and  clear  with  anilin  oil  to  which  a  little  basic  fuchsin 

FIG.  69. 


Actinomycosis  fungus  in  pus.    Fresh,  unstained  preparation.    Magnified 
about  500  diameters.    (Abbott.) 

has  been  added,  and  mount  in  xylol  balsam.  In  this  way 
the  cocci  in  the  centre  and  the  threads  of  the  mycelium  are 
stained  blue  ;  the  club-like  extremities  are  stained  red. 

Biologic  Characters. — The  Streptothrix  actinomyces  grows  in 
all  the  ordinary  artificial  media,  but  from  the  fact  that  all 
the  mycelia  seen  under  the  microscope  are  not  living,  it  is 
often  necessary  to  make  several  cultures  before  obtaining  a 
satisfactory  one.  It  grows  both  with  and  without  oxygen, 
either  at  the  room  temperature  or  at  the  temperature  of  the 
incubator,  and  is  not  resistant  to  heat,  being  destroyed  by  a 
temperature  of  75°  C.  in  five  minutes. 


188  PATHOGENIC  MICROORGANISMS. 

Its  growth  on  blood-serum  and  agar  is  as  isolated  colonies 
on  the  surface  of  these  media.  The  colonies  are  yellowish 
red  and  covered  with  a  sort  of  fluffy  down.  After  a  few 
weeks  the  colonies  run  together  and  form  a  thick  wrinkled 
mass  which  sinks  into  the  media. 

On  gelatin  the  growth  causes  slow  liquefaction.  On  potato 
the  colony  is  yellowish  red  and  limited  in  extent,  has  a  viscid, 
membranous  appearance,  and  is  slow  in  progress.  In  bouillon 
it  causes  no  clouding,  but  develops  on  the  surface  of  the 
medium  as  a  distinct  granular  growth  forming  a  membranous 
film,  which  afterward  sinks  to  the  bottom  of  the  tube. 

Pathogenesis. — Cattle  are  the  most  frequently  affected 
animals,  though  the  disease  has  been  seen  in  swine,  dogs,  and 
horses.  The  common  location  of  the  disease  is  in  the  jaw. 
It  is  not  transmissible  from  animal  to  man.  Inoculations  of 
pure  cultures  are  negative,  though  some  observers  have  suc- 
ceeded by  intravascular  inoculations  in  producing  tumors  from 
which  the  fungus  was  obtained  in  pure  cultures. 

The  disease  is  sometimes  quite  prevalent  among  animals,  and 
appears  to  result  from  the  ingestion  of  vegetable  products  which 
contain  the  streptothrix. 

In  the  earliest  stages  the  parasites  give  rise  to  small  tumors 
resembling  tuberculous  growths,  and  as  these  reach  a  larger 
size  there  is  proliferation  of  the  surrounding  connective 
tissue.  The  tumors  are  then  very  hard,  resembling  osteosar- 
comata.  Suppuration  finally  takes  place,  due  to  the  action 
of  the  fungus  itself,  or  more  probably  to  secondary  infection 
of  the  tumor  by  pus-producing  organisms. 

The  Other  Pathogenic  Streptothrices. 

The  streptothrix  of  madura  foot,  described  by  Wright,  in 
the  Journal  of  Experimental  Medicine,  1898.  and  the  Strepto- 
thrix farcinicus,  discovered  by  Nocard  in  1888,  in  a  disease 
of  cattle  resembling  farcy  in  horses  ;  the  Streptothrix  Eppin- 
geri,  discovered  by  Eppinger  in  a  case  of  acute  abscess  of  the 
brain  ;  and  the  Streptothrix  pseudotuberculosa,  described  by 
Flexner  in  1897,  are  of  interest,  and  the  reader  is  referred 
to  larger  works  on  Bacteriology  for  their  description. 


MALARIA.  189 

MALARIA. 
Plasmodium  Malarise. 

History. — In  1880  Laveran  discovered  in  the  blood  of  cases 
of  intermittent  fever  a  microorganism  which  he  believed  to 
be  the  cause  of  this  disease.  This  microorganism  belongs  to 
the  animal  kingdom  of  the  family  of  the  protozoa,  and  has 
received  a  number  of  names.  The  one  generally  applied 
to-day  is  that  of  Plasmodium  malarise. 

Later  investigations  have  shown  that  this  protozoa  has  two 
cycles  of  existence.  One  cycle  is  reproduced  in  man,  and  the 
other  cycle  in  the  body  of  some  insects  of  the  mosquito  tribe, 
the  Anopheles  claviger  or  Anopheles  maculapennis. 

In  the  red  blood-corpuscles  of  man  the  parasites  go 
through  an  undetermined  number  of  life-cycles,  and  then 
pass  into  the  middle  intestine  of  certain  species  of  mosquitoes, 
in  which  they  go  through  the  various  phases  of  a  new  life- 
cycle,  ending  in  the  salivary  glands,  and  from  these  when  the 
mosquito  bites  a  human  being,  in  order  to  obtain  nourish- 
ment, the  parasite  passes  again  into  man. 

The  phase  of  life  which  is  completed  in  man  is  the  cause  of 
malarial  fever.  In  the  younger  stages  the  parasites  in  this 
life-cycle  appear  as  very  small  amoeboid  bodies,  which  have 
a  more  or  less  rapid  motion,  and  which  are  found  in  the  red 
blood-corpuscles,  nourishing  themselves  with  the  substance  of 
these  corpuscles  and  converting  their  haemoglobin  into  a 
black  pigment  known  as  melanin.  They  increase  in  size, 
cease  their  motion,  and  by  a  process  of  fission  multiply ;  the 
daughter  cells  resulting  from  this  fission  become  free  in  the 
plasma  and  invade  other  blood-corpuscles,  in  which  they  go 
through  the  same  life-cycle. 

The  two  principal  symptoms  of  malaria,  anaemia  and  inter- 
mittent fever,  are  related  to  this  life-cycle,  the  anaemia  being 
due  to  the  destruction  of  a  large  number  of  the  red  blood- 
corpuscles  by  the  parasites,  and  the  fever  is  manifested  when 
the  parasite  is  undergoing  multiplication. 

In  their  growth  the  parasites  of  malaria  have  been  shown 


190  PATHOGENIC  MICROORGANISMS. 

to  belong  to  different  varieties,  differing  in  their  appearance, 
their  mode  of  division,  and  the  length  of  time  which  they 
take  to  go  through  their  whole  life-cycle.  Three  varieties 
are  at  present  recognized  :  the  tertian,  which  goes  through  its 
life-cycle  in  man  in  forty-eight  hours ;  the  quartan,  whose 
life-cycle  is  three  days  ;  and  the  aestivo-autumnal,  whose  life- 
cycle  is  indefinite  and  irregular. 

These  different  species  have  constant  characteristics,  and 
are  not  transformed  from  one  into  another,  though  Laveran 
claims  that  they  are  modifications  of  one  and  the  same 
species,  and  are  interchangeable.  Recent  researches  have 
demonstrated  that  each  variety  of  malaiial  parasite  is  the 
cause  of  a  particular  kind  of  malarial  fever,  and  by  a  micro- 
scopic examination  of  the  blood  of  a  patient  one  may  authori- 
tatively state  the  kind  of  malaria  with  which  that  patient  is 
affected. 

In  addition  to  the  life-cycle  which  begins  and  ends  in  the 
human  subject,  there  is  another  one  which  only  begins  in  man. 
For  instance,  some  of  the  parasite  bodies  increase  in  size  ;  they 
do  not  divide,  but  getting  free  in  the  blood-plasma  they  are 
found  as  bodies  of  characteristic  shape,  larger  than  the  red 
blood-corpuscles.  These  bodies  circulate  in  the  blood  for  a 
number  of  days,  not  giving  rise  to  any  phenomena  ;  and  if  they 
remain  sterile,  they  degenerate  and  disappear.  Upon  exam- 
ination it  is  found  that  some  of  those  bodies  throw  off  flagella 
which  move  with  great  rapidity  among  the  red  blood-cor- 
puscles ;  others  do  not  present  this  phenomenon.  In  the 
aestivo-autumnal  parasites,  on  account  of  their  appearance 
these  bodies  have  been  called  crescent  bodies.  Some  persons 
regard  these  forms  as  degenerated  forms ;  but  it  has  been 
definitely  ascertained  that  those  bodies  which  degenerate  and 
disappear,  when  they  remain  in  man  are  capable  in  the  intes- 
tines of  certain  species  of  mosquitoes  of  starting  a  second 
life-cycle  which  may  be  described  as  follows  :  When  a  mos- 
quito of  the  Anopheles  variety  bites  a  person  in  whose  blood 
these  crescent  bodies  are  present,  or  their  analogous  form  in 
the  other  species  of  parasites,  some  of  them  are  taken  up 
with  the  blood  and  lodge  in  the  mid-intestine  of  the  mos- 


MALARIA.  191 

quito.  Certain  of  these  crescent-forms  give  out  flagella 
which  are  motile  filaments  containing  chromatin,  and  these 
loose  filaments  penetrate  and  fecundate  other  crescent-forms. 
And  the  fecundated  bodies  are  after  this  able  to  penetrate  the 
epithelium  of  the  intestines  and  travel  between  the  muscular 
fibres  of  the  intestines.  There  is  therefore  a  differentiation  of 
.sv.r  hctween  the  crescent  bodies :  those  becoming  flagellated  are 
the  male  elements,  the  microgametocytes ;  and  the  others, 
which  do  not  become  flagellated,  are  macrogametes,  the  fe- 
male. These  macrogametes  after  fecundation  develop  between 
the  muscles  of  the  intestines  of  the  mosquito,  becoming  sur- 
rounded with  a  capsule  and  acquiring  the  characteristics  of 
typical  sporozoa.  After  a  while  its  nucleus  divides  into  a 
number  of  smaller  ones,  which  in  their  turn  become  the 
nucleus  of  the  dividing  cell  itself,  the  sporozoite.  These 
sporozoites  are  set  free  by  rupture  of  the  capsule  of  the 
sporozoa,  and  are  scattered  throughout  the  body  of  the  mos- 
quito, some  finding  lodgement  in  the  tubules  of  the  salivary 
glands,  and  when  the  insect  again  stings  man,  sporozoites  are 
inoculated  into  him  together  with  the  irritating  secretion  of 
the  gland. 

This  cycle  lasts  in  the  mosquito  from  eight  to  ten  days,  and 
varies  with  the  species  of  the  parasite.  It  is  likely  that  this 
represents  the  whole  life  of  the  malarial  parasite,  and  it  has 
been  demonstrated  that  the  infected  mosquito  does  not  trans- 
mit the  malarial  parasite  to  its  larva,  the  two  life-cycles  in 
man  and  the  mosquito  being  sufficient  to  explain  the  known 
practical  facts. 

The  three  varieties  of  the  malarial  parasites  found  in  the 
human  blood  ditfer  as  to  size,  distribution  of  their  pigments, 
in  the  number  of  daughter  cells  produced  from  one  parasite, 
and  the  length  of  time  required  for  the  completion  of  their 
life-cycle.  One  form,  the  tertian,  requires  forty-eight  hours ; 
another,  the  quartan,  requires  seventy-two  hours;  and  a 
third  form,  the  sestivo-autumnal,  has  an  indefinite  life-cycle. 
Occasionally  there  are  seen  what  are  known  as  the  double- 
tertian  and  double-quartan  forms  of  malarial  fever,  in  which 
the  fever  is  produced  by  several  generations  of  one  of  those 


192  PATHOGENIC  MICROORGANISMS. 

two  forms  of  parasites  going  through  their  life-cycles  begin- 
ning at  different  times  and  on  successive  days,  so  that  the 
fever  has  the  appearance  of  a  quotidian  form  of  fever.  The 
three  different  varieties  of  parasites  will  be  best  understood 
by  referring  to  the  figures  on  Plate  VI. 

Examination  of  the  Blood  of  Man  for  Diagnostic  Purposes.-— 
The  following  two  methods  always  serve  best :  (1)  the  fresh 
blood  examination  and  (2)  the  examination  of  stained  speci- 
mens. Whenever  practicable,  fresh  blood  examination  offers 
the  easiest  and  best  method  for  diagnosis. 

The  technic  is  as  follows :  Thoroughly  clean  cover-glasses 
and  slides,  being  careful  to  remove  all  greasy  matter.  Cleanse 
also  the  skin  of  the  lobe  of  the  ear  or  tip  of  a  finger,  make 
an  incision  with  a  sharp-pointed  knife,  wipe  off  the  first 
exuding  drop,  touch  the  top  of  the  next  drop  with  a  clean 
cover-glass  held  with  forceps,  being  careful  to  avoid  touching 
the  skin,  and  taking  the  drop  when  small  so  that  the  corpus- 
cles will  be  spread  out  in  a  uniform  layer,  not  in  rouleaux, 
when  the  cover-glass  is  laid  on  the  slide ;  press  the  cover- 
glass  on  to  the  slide  gently ;  if  the  cover-glass  and  slide  are 
clean,  the  blood  will  spread  in  an  even  thin  layer ;  examine 
with  a  Y1^  oil-immersion.  Preparations  made  in  this  way  will 
show,  if  examined  immediately,  the  amoeboid  plasmodium 
inside  of  the  red  blood-cells,  being  especially  recognizable  by 
the  movements  or  the  contained  pigment.  It  is  possible 
sometimes  to  keep  such  preparations  for  several  hours. 

When  examination  of  fresh  blood  is  not  practicable,  or 
when  it  is  desired  to  preserve  the  preparation,  resort  to  the 
procedure  of  staining  must  be  had.  This  is  best  accomplished 
by  the  methylene-blue  and  eosin  methods,  either  applied 
together,  or  each  dye  being  used  separately,  as  follows  : 

A  drop  of  blood  is  taken  as  just  described,  spread  evenly  upon 
a  thin  cover-glass,  and  allowed  to  air-dry ;  the  film  is  then 
set  by  immersing  the  cover-glass  for  twenty  to  thirty  minutes 
in  a  mixture  of  equal  parts  of  absolute  alcohol  and  ether : 
after  drying,  the  mixed  stain  (methylene-blue  and  eosin)  is 
applied  over  the  surface  of  the  film  and  allowed  to  remain  for 
five  minutes ;  it  is  then  poured  off,  the  preparation  washed 


PLATE  VI 


*:• 


FIGS,  i,  2,  and  3  show  three  phases  of  the  parasite  of  tertian  fever. 
Fig.  i,  ring  form,  showing  beginning  pigment  formation.  Ing.  2,  full- 
grown  parasite.  Fig.  3,  segmenting  bodies.  (WELCH  and  TH AVER.) 

FIGS.  4,  5,  and  6  show  the  parasite  of  quartan  fever  at  different  stages  of 
growth.  Fig.  4,  moderately  developed  iutracorpuscular  parasite.  Fig.  5. 
large  swollen  extracorpuscular  form.  Fig.  6,  flagellate  body.  (WELCH 
and  THAYER.) 

FIGS.  7,  8,  and  9  illustrate  the  sestivo-autumnal  parasite.  Fig.  7,  ring- like 
body,  with  a  few  pigmented  granules.  Fig.  8,  crescent  still  in  blood-cor- 
puscle. Fig.  9,  vacuolation  of  crescent.  (WELCH  and  THAYER.) 

FIG.  10.  Amoeba  from  section  of  intestine  hardened  in  alcohol  and 
stained  with  methylene  blue.  (COUNCILMAN  and  L/AFLEUR.) 


MALARIA.  193 

thoroughly  in  distilled  water  three  or  four  times,  then  dried 
and  mounted  in  balsam,  and  examined.  In  this  preparation 
the  red  cells  will  be  stained  pinky  the  leucocytes  pale  blue  and 
their  nuclei  dark  blue  ;  the  parasites  will  be  stained  very  dark 
blue  and  their  pigment-granules  remain  unstained. 

The  mixture  of  methylene-blue  and  eosin  is  prepared  as 
follows : 

Saturated  aqueous  solution  of  methyl-blue,  1  part; 
Alcoholic  solution  of  eosin  (1  per  cent.),     2  parts. 
Do  not  filter. 

Loeffler's  methylene-blue,  and  Ehrlich's  hsematoxylin  and 
eosin,  give  also  at  times  very  excellent  preparations. 

The  varieties  of  plasmodia  may  be  distinguished  from  each 
other. 

Differentiation  between  the  Tertian  and  Quartan  Parasites : 

1st.  The  tertian  parasite  completes  its  life-cycle  in  two 
days ;  that  of  the  quartan  requires  three  days. 

2d.  The  tertian  parasite  has  a  tendency  to  discolor  the 
blood-cells  and  to  enlarge  them.  The  quartan  does  not  dis- 
color so  much,  and  never  enlarges  its  containing  red  cell ;  on 
the  contrary,  the  cell  generally  appears  smaller. 

3d.  The  quartan  parasite  has  better-defined  and  clearer 
outlines  and  coarser  pigment-granules  than  the  tertian. 

4th.  In  fission  the  quartan  parasite  divides  into  six  to 
twelve  daughter  cells,  which  are  larger  and  contain  each  a 
refractive  granule  in  its  centre.  The  tertian  parasite  divides 
into  fifteen  to  twenty  daughter  cells,  smaller,  and  with  no 
central  granule. 

Inoculation. — Blood  infected  with  either  kind  of  parasite 
when  inoculated  into  healthy  individuals  has  in  a  number  of 
'•uses  produced  that  variety  of  fever  specific  for  the  particu- 
lar parasite,  and  this  special  parasite  has  been  found  in  the 
blood  of  the  inoculated  individual. 


13— M.  B. 


194  PATHOGENIC  MICROORGANISMS. 

AMOEBIC  COLITIS. 
Amoeba  Coli. 

History. — In  certain  forms  of  chronic  dysentery  accom- 
panied with  ulcerations  of  the  lower  bowel,  and  which  very 
often  give  rise  to  suppuration  in  internal  organs,  especially 
the  liver,  an  animal  parasite,  the  Amoeba  coli,  has  been  accu- 
rately described  since  1875  by  Losch,  of  St.  Petersburg. 
Losch's  observations  have  been  confirmed  since  by  a  number 
of  other  observers. 

Morphology. — The  amoeba  is  a  protozoa,  and  consists  of 
protoplasm  which  exhibits  under  different  conditions  various 
forms.  In  the  quiescent  condition  it  is  spherical  in  shape, 
and  may  be  recognized  from  the  other  cellular  elements  by 
its  greater  refraction  of  light  and  by  its  pale-green  color.  Its 
size  is  from  10  to  25  mikrons.  The  body  consists  of  two 
parts  :  an  inner  part,  or  endoplasm,  which  is  generally  gran- 
ular and  of  dark  color ;  and  an  outer  part,  or  ectoplasm,  which 
is  pale  and  white.  These  two  parts  may  be  best  made  out  in 
the  motile  amoeba.  A  nucleus  more  or  less  central  is  also 
easily  made  out. 

The  Amoeba  coli  is  stained  easily  by  any  of  the  nucleolar 
stains,  especially  by  methylene-blue.  In  its  body  may  often 
be  seen  foreign  bodies,  especially  red  blood-cells,  but  it  rarely 
contains  leucocytes  or  fat. 

The  motion  of  the  amoeba  is  caused  by  the  mechanism  of 
pseudopodia,  which  are  blunt  homogeneous  processes,  the  pro- 
trusion of  a  portion  of  the  ectoplasm.  Motion  is  sometimes 
gradual  and  deliberate,  at  other  times  rapid,  and  is  modified 
by  variations  in  temperature. 

Biology. — Nothing  is  known  as  to  the  functions  of  nutri- 
tion, respiration,  and  reproduction  of  the  amoeba.  It  is  found 
occasionally  in  health  in  the  secretions  of  the  lower  bowel, 
and  in  cases  of  dysentery  may  disappear  partially  or  com- 
pletely from  the  stools  during  convalescence.  They  are  also 
frequently  found  in  the  pus  of  hepatic  abscesses. 

Examination  of  the  feces,   especially  the  slimy  portion  of 


QUESTIONS.  195 

dysenteric  stools,  and  the  pus  in  cases  of  suspected  amoebic 
infection,  should  be  made  in  fresh  specimens,  when  the  live 
amoebae  may  be  easily  recognized.  Dried  preparations  are 
made  as  for  bacterial  examination,  and  colored  with  aqueous 
solution  of  methylene-blue. 


QUESTIONS. 
Streptothrix. 

What  is  a  Streptothrix  ?  How  does  it  resemble  moulds  ?  How  does  it  dif- 
fer from  moulds?  How  does  it  resemble  bacteria? 

Which  are  the  best  known  of  the  streptothrices  ? 

Why  are  the  Bacillus  tuberculosis  and  Bacillus  diphtherix  believed  to  belong 
to  the  class  of  the  streptothrices  ? 

What  is  the  appearance  of  the  lesion  caused  by  streptothrices  ? 

Describe  the  Streptothrix  actinomyces,nT  ray  fungus.  How  is  it  best  stained? 

Give  Mallory's  staining  method. 

Give  the  principal  biologic  characters  of  the  Streptothrix  actinomyces. 
How  does  it  grow  on  blood-serum  and  agar  ?    On  gelatin  ?    On  potato  ?    In 
bouillon  ? 

What  animals  are  susceptible  to  Streptothrix  infection  ?  Is  the  disease 
transmissible  from  animal  to  animal  or  from  animal  to  man  ?  How  are  ani- 
mals infected  ? 

Describe  the  lesions  produced  by  Streptothrix  infection.  What  causes  the 
suppuration  ? 

What  other  pathogenic  streptothrices  have  been  described  and  studied  ? 


Malaria. 

What  is  the  Plasmodium  malarise  f 

How  many  life-cycles  has  it?    Where  are  those  phases  of  life  completed? 

Describe  the  life-cycle  of  the  plasmodium  in  man. 

What  are  the  two  principal  symptoms  of  malaria,  and  what  relation  have 
thev  to  the  development  of  the  parasite? 

What  differentiates  the  varieties  of  the  malarial  parasites  from  each  other  ? 

What  three  varieties  are  at  present  recognized? 

What  forms  of  fever  are  caused  by  these  different  varieties? 

What  life-cycle  only  begins  in  the  body  of  man? 

Describe  the  flagellated  and  non-flagellated  bodies? 

What  are  the  crescent  bodies? 

How  do  those  crescent  bodies  or  their  homologues  develop  in  the  body  of 
the  mosquito? 

What  are  the  microgametocytes? 

What  are  the  macrogametes  ? 

Describe  the  development  of  the  fecundated  macrogametes  in  the  mos- 
quito ? 

What  are  the  sporozoites,  and  how  do  they  infect  man  ? 

What  is  the  duration  of  the  life-cycle  parasite  in  the  mosquito? 

Does  the  mosquito  transmit  the  malarial  parasite  to  its  larva? 


196        EXAMINATIONS  OF   WATER,  AIR,  AND  SOIL. 

What  is  meant  by  the  double-tertian  and  double-quartan  forms  of  mala- 
rial fever  ? 

To  what  is  the  quotodian  form,  of  fever  due  ? 

Give  the  technic  for  the  examination  of  fresh  malarial  blood  for  diag- 
nostic purposes. 

How  are  stained  specimens  prepared  for  examination  ? 

What  is  the  differentiation  between  the  quartan  and  tertian  parasite  in 
the  blood  ? 

Is  the  blood  of  malarial  patients  infectious  ? 

Amoeba  Coli. 

Where  is  the  Amoeba  coli  found?  To  what  kingdom  does  it  belong? 
Describe  it. 

What  is  the  endoplasm  ? 

What  is  the  ectoplasm  ? 

How  is  Amoeba  coli  stained? 

What  may  be  seen  in  the  body  of  the  Amoeba  coli  ? 

What  causes  the  motion  of  the  amoeba  ? 

How  is  examination  for  the  Amoeba  coli  practised  ? 


CHAPTER    XXIV. 

BACTERIOLOGICAL    EXAMINATIONS    OF    WATER,    AIR, 
AND  SOIL. 

THE  BACTERIOLOGICAL  INVESTIGATION  OF  WATER. 

BOTH  qualitative  and  quantitative  bacteriological  examina- 
tions of  water  are  often  resorted  to  in  order  to  test  the  adapta- 
bility of  this  substance  for  human  and  animal  consumption. 
By  the  quantitative  test  the  number  of  bacteria  present  in 
the  water  is  ascertained  without  any  reference  to  their  patho- 
genic character,  and  when  this  number  exceeds  500  bacteria 
per  c.c.  the  water  is  condemned.  This  is  evidently  not  a 
very  fair  test,  but  it  is  not  without  value  when  it  is  consid- 
ered that  in  a  number  of  instances  the  virulence  of  some 
pathogenic  microorganisms  is  increased  when  they  are  inocu- 
lated together  with  saprophytic  germs ;  and  again,  that  the 


BACTERIOLOGICAL  INVESTIGATION  OF  WATER.    197 

introduction  of  non-pathogenic  bacteria  occasionally  so  dimin- 
ishes the  animal  resistance  that  animals  ^resistant  to  inocula- 
tions by  some  pathogenic  bacteria  are  subsequently  rendered 
susceptible. 

This  quantitative  analysis  is  especially  useful  in  cases  in 
which  the  mean  quantity  of  bacteria  in  a  given  body  of 
water  is  already  known,  and  as  a  matter  of  comparison  to 
ascertain  whether  any  new  source  of  contamination  has  been 
introduced. 

The  examination  is  made  as  follows :  A  sample  of  the 
water  is  collected  in  clean  sterilized  bottles  or  tubes.  If  the 
water  is  from  a  pump,  well,  or  from  a  cistern,  it  should  be 
allowed  to  run  for  a  few  minutes  before  the  sample  is  taken. 
If  the  water  is  from  a  spring,  river,  or  any  collection  of 
water,  the  sample  for  examination  should  be  taken  a  foot 
or  two  below  the  surface  of  the  water.  Agar  and  gelatin 
plates  should  be  immediately  inoculated  with  the  water. 
When  this  is  not  practicable  the  plates  should  be  made  at 
as  early  a  time  as  possible,  the  samples  meanwhile  being 
kept  on  ice,  near  the  freezing-point,  to  prevent  the  further 
development  of  bacteria. 

For  the  purpose  of  collecting  water  for  examination,  glass 
bulbs  after  the  pattern  of  Sternberg  (Fig.  70)  are  very  use- 
ful. These  consist  of  a  sphere  blown  on  the  end  of  a  glass 

FIG.  70. 


Glass  bulb  for  collecting  samples  of  water.    (Abbott.) 

tube,  the  stem  at  the  other  end  terminating  in  a  capillary 
tube.  After  thoroughly  cleaning  these  bulbs,  a  negative 
vacuum  is  established  therein  by  introducing  in  each  tube 
a  few  drops  of  water,  allowing  same  to  boil  over  a  gas-flame, 
and  when  the  water  has  completely  vaporized  into  steam  the 
capillary  end  of  the  bulb  is  brought  into  the  flame  and  the 
apparatus  sealed.  When  it  is  desired  to  collect  a  sample  of 


198        EXAMINATIONS  OF  WATER,  AIR,  AND  SOIL. 

water,  the  capillary  end  of  the  bulb  is  broken  under  the 
water  and  the  vacuum  in  the  tube  causes  a  suction  of  the 
fluid  into  the  bulb  until  same  is  about  three-fourths  full. 
When  this  is  accomplished,  the  bulb  should  again  be  sealed 
and  packed  in  ice. 

When  making  gelatin  and  agar  plates  a  known  quandty 
of  water  should  always  be  used  to  each  plate,  say  0.01, 
0.05,  0.10,  or  1  c.c.,  the  quantity  varying  according  to  the 
amount  of  suspected  contamination  of  the  water.  The  plates 
are  made  as  already  described  in  the  chapter  on  the  making 
of  plates,  only  plate  No.  1,  however,  being  made.  After 
twenty-four  or  forty-eight  hours  the  colonies  on  the  plate  are 
counted ;  and  as  it  is  assumed  that  each  colony  is  grown 
from  a  single  bacterium,  the  number  of  colonies  on  the  plate 
represents  the  number  of  bacteria  originally  in  the  sample. 
Two  sets  of  plates  should  always  be  made,  one  on  gelatin,  which 
is  kept  at  the  room  temperature,  and  another  on  agar  for  the 
incubator.  When  the  number  of  colonies  on  the  plates  is 
very  large,  plates  should  be  made  with  still  greater  dilution, 
so  as  to  obtain  plates  with  only  a  moderate  number  of  bac- 
teria, in  order  to  facilitate  the  count.  Absolutely  sterile  water 
should  always  be  used  to  make  the  dilution.  In  expressing 
results,  the  number  of  bacteria  in  1  c.c.  of  the  water  is  men- 
tioned. For  counting  the  colonies  on  the  plates  the  counter 
of  Wolfhuegel  (Fig.  31)  is  used,  which  consists  of  a  wooden 
framework,  at  the  bottom  of  which  may  be  put  a  glass  plate, 
white  or  black,  in  order  to  form  a  background  for  the  plate 
containing  the  colonies.  Over  this  background  the  plate  of 
which  the  colonies  are  to  be  counted  is  placed,  and  over  this 
a  transparent  glass  plate  ruled  in  square  centimeters,  and 
held  in  position  just  above  the  colonies,  but  without  touching 
them.  It  is  easy  in  this  way  to  count  the  colonies  in  each 
square  centimeter,  and  so  make  out  the  total  number  of  col- 
onies on  the  plate.  When  this  is  found  too  tedious,  a  number 
of  squares  at  different  portions  of  the  plate  may  be  counted, 
an  average  established,  and  that  average  multiplied  by  the 
number  of  squares  will  give  approximately  the  number  of 
colonies  on  the  plate.  By  multiplying  the  number  of  col- 


BACTERIOLOGICAL  INVESTIGATION  OF  WATER.    199 

onies  on  the  plate  according  to  the  degree  of  dilution  of  the 
water,  it  is  easy  to  arrive  at  the  number  of  bacteria  in  a  cubic 
centimeter  of  the  water.  Thus  if  the  average  number  of  col- 
onies per  square  is  15,  and  there  are  100  squares  on  the  plate, 
and  the  amount  of  water  used  in  making  the  plate  was  0.10 
c.c.,  then  15  X  100  X  10  equals  15,000,  which  represents 
the  number  of  bacteria  present  in  1  c.c.  of  the  water. 

Petri  dishes  may  be  used  instead  of  plates,  and    for  the 
counting  of  colonies  on  the  same  special  means  have  been 


6  8 

Fakes'  apparatus  for  counting  colonies  (reduced  one-third). 

devised.  That  of  Fakes'  apparatus  (Fig.  71)  is  a  cheap  and 
convenient  one.  It  consists  of  a  sheet  of  paper  on  which  is 
printed  a  black  disc  ruled  with  white  lines.  The  Petri  dish 
is  placed  centrally  upon  this  paper,  and  the  colonies  between 
the  white  lines  are  counted,  the  whole  circle  being  divided 
into  sixteen  equal  segments,  as  seen  in  the  figure. 


200       EXAMINATIONS  OF  WATER,  AIR,  AND  SOIL. 

For  counting  colonies,  a  small  hand  lens,  such  as  is  repre- 
sented in  Fig.  72,  is  often  necessary. 


FIG.  72. 


Lens  for  counting  colonies. 

Instead  of  plates  and  Petri  dishes,  Esmarch  tubes  may 
be  used  as  follows  :  A  definite  quantity  of  the  water  is  added 
to  melted  gelatin  in  a  test-tube  and  the  same  rolled  as 
described  previously. 

A  special  counter  for  Esmarch  tubes,  provided  with  a  mag- 
nifying glass,  is  used  in  counting  the  colonies  (Fig.  73). 

FIG.  73. 


Esmarch's  apparatus  for  counting  colonies  in  rolled  tubes.    (Abbott.) 

As  previously  mentioned,  the  value  of  this  quantitative 
examination  of  the  bacteria  of  water  is  not  very  great,  because. 


BACTERIOLOGICAL  INVESTIGATION  OF  WATER.    201 

though  of  great  utility,  it  does  not  give  definite  information 
as  to  the  poisonous  organisms  of  the  water.  For  this  pur- 
pose a  qualitative  examination  for  pathogenic  germs  is  of 
much  more  value.  This  is  not,  however,  as  easily  per- 
formed, and  in  the  great  majority  of  cases  gives  negative 
results.  & 

The  bacteria  most  often  sought  in  this  way  are  the  bacillus 
of  cholera  and  the  bacillus  of  typhoid  fever,  both  of  which 
are  short-lived  in  water,  and  have  only  rarely  been  demon- 
strated, partly  perhaps  on  account  of  their  low  vitality  in 
water,  and  partly  also  because  they  are  looked  for  at  a  date 
considerably  later  than  that  at  which  they  were  originally 
contained  in  the  water.  From  the  very  nature  of  these 
bacteria  it  is  easy  to  understand  why  they  should  be  short- 
lived. The  presence  in  the  water  of  a  number  of  ordinary 
saprophytes  interferes  with  the  growth  of  pathogenic  bacteria, 
and  either  destroys  them  or  consumes  the  pabulum  neces- 
sary for  their  growth.  Sedimentation  of  the  water,  which  is 
constantly  taking  place,  carries  along  with  insoluble  inorganic 
matter  the  bacteria  to  the  bottom ;  and  in  running  water 
the  oxygen  of  the  air  and  direct  sunlight  act  as  efficient 
germicides. 

In  making  qualitative  examinations  of  water  for  typhoid 
and  cholera  germs,  what  has  been  said  in  the  special  chapters 
on  those  germs  should  be  borne  in  mind.  It  is  advan- 
tageous, for  instance,  to  mix  the  water  with  three  times  its 
volume  of  sterile  bouillon,  and  to  incubate  this  mixture  before 
making  the  plates. 

For  cholera,  after  six  hours  in  the  incubator  the  plates  may 
be  made,  taking  for  this  purpose  the  fluid  from  the  upper 
portion  of  the  mixture,  as  this  germ  grows  rapidly,  and 
chiefly  on  the  surface  of  the  fluid. 

For  typhoid  fever  the  incubation  should  be  continued  for 
two  or  three  days  before  making  the  plates.  By  this  incu- 
bation the  ordinary  saprophytes  are  retarded  in  growth,  as 
they  have  less  tendency  to  thrive  at  the  incubator  tempera- 
ture, whereas  the  reverse  is  the  case  for  pathogenic  germs, 
which  grow  much  more  rapidly  at  37°  C.,  and  are  thus  in 


202        EXAMINATIONS  OF  WATER,  AIR,  AND  SOIL. 

relatively  larger  numbers.  Two  sets  of  plates  should  be  made 
as  described  in  the  quantitative  test,  one  to  be  kept  at  the 
room  temperature  and  the  other  to  be  incubated,  and  after 
these  have  grown  for  twenty-four  to  forty-eight  hours  the 
colonies  should  be  examined  under  a  low  power  of  the  micro- 
scope, and  the  colonies  picked  up  with  a  platinum  needle  and 
planted  in  fresh  agar  and  gelatin  tubes,  and  these  plated 
again  until  pure  cultures  are  obtained. 

Eisner's  method,  described  in  the  chapter  on  typhoid  fever, 
is  a  very  good  method  for  the  separation  of  water  bacteria 
from  typhoid  bacteria,  and  the  reader  is  referred  to  that 
chapter  for  its  description. 

The  addition  of  antiseptics,  in  small  amounts,  helps  in  the 
recognition  of  pathogenic  germs,  such  as  typhoid,  in  water, 
as  these  antiseptics  interfere  more  materially  with  the  growth 
of  saprophytes  than  they  do  with  that  of  the  pathogenic  germs. 

The  reader  is  again  reminded  that  the  isolation  of  typhoid 
fever  germs  from  water  is  difficult. 

It  is  easier  to  examine  water  for  the  Bacillus  coli  communis, 
which  when  present  shows  contamination  by  animal  or 
human  excreta,  and  makes  the  water  unfit  for  consumption. 

By  inoculating  glucose-  or  lactose-bouillon  in  fermentation- 
tubes,  the  presence  of  this  bacillus  is  easily  made  out  on  ac- 
count of  the  rapid  development  of  gases  it  produces ;  and 
plates  made  from  the  bouillon  in  the  closed  arm  of  the 
fermentation-tube  will  yield  in  such  cases  almost  pure  cult- 
ures of  this  bacillus. 

BACTERIOLOGICAL  EXAMINATION  OF  THE  AIR. 

A  number  of  methods  have  been  suggested  for  this  pur- 
pose ;  some  consist  in  exposing  plates  of  nutrient  media,  such 
as  gelatin  and  agar,  in  the  air,  the  bacteria  falling  on  these 
plates  and  developing ;  or,  again,  causing  by  slow  aspiration 
a  measured  volume  of  air  to  pass  through  substances,  such  as 
sterilized  sand  or  granulated  sugar,  afterward  making  agar 
and  gelatin  plate  cultures  with  them.  Quiet  air  contains  but 
few  bacteria,  but  air  in  motion,  as  in  blowing  wind,  carries 


BACTERIOLOGICAL  EXAMINATION  OF  THE  SOIL.  203 

solid  substances,  such  as  dirt,  etc.,  loaded  with  bacteria.  These 
may  travel  in  suspension  in  the  air  for  a  considerable  distance. 

The  Sedgwick-Tucker  method  is  perhaps  the  best  procedure 
for  the  examination  of  air. 

For  this  purpose  an  apparatus  known  as  the  aerobioscope 
(Fig.  74)  is  required.  In  this  apparatus  a  certain  amount  of 
sterile  dry  granulated  sugar  is  introduced  into  the  narrow 
part  of  the  tube  at  d ;  and  at  a  a  small  roll  of  fine  brass 
wire-gauze  is  inserted  in  order  to  provide  a  stop  for  the 
filtering  material  which  is  placed  over  it,  as,  for  example, 
the  sugar;  then  by  means  of  an  air-pump  a  certain  defi- 
nite quantity  of  air  is  sucked  through  the  aerobioscope, 
after  which  the  apparatus  is  closed  with  sterile  cotton  at 

FIG.  74. 


d  a  b 

The  Sedgwick-Tucker  aerobioscope.    (Abbott.) 

b  and  at  c,  and  by  gentle  tapping,  the  contaminated  sugar 
is  forced  into  the  larger  portion  of  the  tube  at  e;  when 
this  is  accomplished  20  c.c.  of  liquefied  sterile  gelatin  are 
poured  in  the  larger  part  of  the  tube,  the  sugar  dissolved  in 
this  gelatin,  and  an  ordinary  Esmarch  tube  made.  The 
colonies  may  be  counted  as  in  an  Esmarch  tube,  and  pure 
cultures  of  the  isolated  colonies  may  be  made  on  other  plates. 

THE  BACTERIOLOGICAL  EXAMINATION  OF  THE  SOIL. 

In  the  study  of  the  soil  for  microorganisms  special  instru- 
ments for  collecting  the  soil  at  different  depths  have  been  in- 
vented. C.  Fraenkel's  apparatus  is  perhaps  the  most  useful. 

Small  fragments  of  the  soil  to  be  examined  should  be  dis- 
solved in  liquid  gelatin  or  agar,  plates  made,  and  the  colonies 
counted  as  for  the  examination  of  water. 

The  objection  to  this  method,  however,  lies  in  the  fact  that 


204       EXAMINATIONS  OF  WATER,  AIR,    AND  SOIL. 

the  solid  particles  of  earth  interfere  considerably  with  the 
counting  of  the  colonies,  and  to  obviate  this  it  is  necessary  at 
times  to  dissolve  the  soil  in  a  certain  quantity  of  sterile 
water,  and  to  make  plate  cultures  from  this  water. 

It  should  always  be  remembered,  however,  in  making 
examinations  of  the  soil,  that  a  number  of  the  bacteria  found 
in  it  are  anaerobics,  and  should  be  cultivated  as  such. 

Soil  taken  near  the  surface  is  always  rich  in  bacteria,  and 
the  further  down  the  investigator  proceeds  the  smaller  is  the 
number  of  bacteria  found,  until  at  a  distance  of  a  meter  and 
a  half  from  the  surface  all  bacteria  have  disappeared. 

In  examinations  of  excavations  made  in  the  city  of  New 
Orleans  some  three  years  ago  the  author  had  occasion  to 
verify  the  foregoing  fact  fully ;  cultures  made  from  mud  at 
different  depths  showed  a  constant  diminution  of  micro- 
organisms, until  at  a  depth  of  between  five  and  six  feet 
no  bacteria  could  be  obtained. 

QUESTIONS. 

What  sort  of  bacteriological  examinations  of  water  are  made? 

What  is  meant  by  a  quantitative  test  ? 

Why  is  this  not  a  fair  test? 

How  is  a  quantitative  analysis  of  water  made  ? 

What  instrument  is  useful  for  that  purpose  ? 

How  should  water  be  collected  ? 

What  dilutions  should  be  used  ? 

How  is  this  dilution  made  ? 

How  are  the  colonies  on  plates  counted? 

Describe  Wolfhuegel's  apparatus  for  counting  colonies  on  plates? 

How  are  Petri  dishes  used  ? 

Describe  Fakes'  apparatus  for  counting  colonies  in  Petri  dishes. 

How  are  Esmarch  tubes  made  ? 

For  what  bacteria  is  the  qualitative  analysis  of  water  made  ? 

What  are  the  difficulties  in  making  qualitative  analysis? 

How  is  examination  of  water  for  the  cholera  germ  made  ?  For  typhoid 
fever  germ  ? 

Describe  Eisner's  method  of  examining  water  for  typhoid  fever  bacilli. 

What  influence  has  the  addition  of  antiseptics  to  water  for  bacterial  exam- 
ination? 

How  is  water  to  be  examined  for  the  presence  of  Bacillus  coli  communis  ? 

What  is  the  significance  of  the  Bacillus  coli  communis  in  water? 

Describe  a  method  for  the  bacteriological  examination  of  air. 

Describe  Sedgwick-Tucker's  method. 

How  is  an  examination  of  the  soil  made  ? 

What  bacteria  are  found  in  the  soil  ? 

At  what  depth  from  the  surface  is  the  soil  free  from  bacteria  ? 


INDEX. 


ABBE  condenser,  20 
Abdominal    contents,    examina- 
tion of.  89 

Abstraction  theory,  97 
Acquired  immunity,  95 
Actinomyces,  186 

biologic  characters  of,  187 
Mallory's  stain  of,  187 
morphology  of,  186 
staining  of,  187 
Actinomycosis,  186 
diagnosis  of,  188 
history  of,  186 
pathogen  esis  of,  188 
Active  immunity,  95 
Aerobic  bacteria,  36 
Agar,  filtration  of,  60 

media,  59 
Agglutination  of  M.  melitensis,  114 

serum,  l&l 
Agglutinin,  162 
Air,  202 

bacteriological  examination  of,  202 
Sedg  wick-Tucker  method,  203 
Amosba  coli,  194 
biology  of,  194 
morphology  of,  194 
motion  of,  194 
staining  of,  194 
colitis,  194 

history  of,  194 
Amphitrocha,  35 
Anaerobic  bacteria,  36 
cultivation  of,  72 
Animals,  inoculation  of,  85 

observation  of,  89 
Anterior   chamber,    inoculation   into, 

88 

Anthrax,  128 
bacillus  of,  128 

biologic  characters  of,  129,  130 
morphology  of,  128 
pathogeuesis  of,  131 


Anthrax,  bacillus  of,  resistance  of,  to 

thermal  changes,  130 
staining  of,  129 

history  of,  138 

immunization  of,  131,  132 
Antimicrobic  blood-serums,  97 
Antisepsis,  methods  of,  83 
Antiseptics,  81,  83 
Antitoxic  blood-serums,  96 
Antityphoid  serum,  165 
Arnold  steam  sterilizer,  80 
Arthrospore,  34 
Attenuation,  methods  of,  93 
Autopsy  on  animals,  89 
Axial  point,  24 

PACILLUS,  29 

D  authracis,  128 

symptomatica,  153 

biologic  characters  of,  153 
history  of,  153 
immunity  from,  155 
morphology  of,  153 
pathogeuesis  of,  154,  155 
spores  of,  153 
staining  of,  153 
choleras  gallinarum,  183 
biology  of,  183 
inoculation  of,  183 
pathogen  esis  of,  183 
staining  of,  183 
coli  communis,  107,  165 

biologic  characters  of,  166 
etiologic  relations  of,  166 
history  of,  165 
morphology  of,  166 
pathogen  esis  of,  167 
cunicucilidi  (Fluegge),  184 
diphtheria,  133 
dysentericse,  180 
biologic  characters  of,  181 
history  of,  180 
inoculation  of,  181,  182 

205 


206 


INDEX. 


Bacillus  dysenteries,  morphology  of, 

180 
leprse,  120 

biology  of,  121 

distribution  of,  120 

history  of,  120 

inoculation  of,  121 

morphology  of,  120 

staining  of,  121 
mallei,  124 

biology  of,  124,  125 

inoculation  of,  126 

morphology  of,  124 

spores  in,  124 

staining  of,  126 
pestis,  176 

biologic  characters  of,  176 

immunity  from,  178 

morphology  of,  176 

pathogenesis  of,  177 

vitality  of,  177 
pneumonise  (Fluegge),  see  Pneumo- 

coccus. 
pseudodiphtherise,  140 

staining  of,  141 

varieties  of,  141 
pyocyaneus,  99,  106 

biologic  characters  of,  106 

morphology  of,  106 

pathogenesis  of,  106 
pyogenes  foetid  us,  106 
morphology  of,  106 
staining  of,  106 
sui  pestifer,  182 

biologic  characters  of,  182 
history  of,  182 
immunity  from,  182 
morphology  of,  182 
pathogenesis  of,  182 
vitality  of,  182 

of  rabbit  septicaemia  (Koch),  184 
of  swine  plague  (Loeffler),  184 
of  syphilis,  122 

staining  of,  123 
tetani,  145 

biologic  characters  of,  146,  147 

morphology  of,  146 

rnotility  of,  148 

pathogenesis  of,  149 

spores  of,  146 

staining  of,  146 

thermal  death- point  of,  148 
tetanus,  cultivation  of,  73 
tuberculosis,  99,  107,  115 

Koch's  discovery  of,  115 


Bacillus  tuberculosis,  morphology  of, 

115 

nature  of,  116 
occurrence  of,  116 
pathogenesis  of,  117 
staining  of,  116 
transmission  of,  118 
typhosus,  99,  107,  156 
artificial  susceptibility  of,  162 
biologic  characters  of,  157 
comparison  with  Bacilli  coli,  159 
cultures  of,  158,  161 
differentiation     of,     from    allied 

groups,  159  et  seq. 
history  of,  156 
inoculation  with,  158 
morphology  of,  156 
occurrence  of,  156 
serum  diagnosis  of,  162 
staining  of,  157 
vitality  of,  158 
varieties  of,  29 
Bacteria,  anaerobic,  72 
cultivation  of,  55 
definition  of,  28 
destruction  of,  81 
examination  of,  41 
isolation  of  (Koch's),  69,  70 
morphology  of,  29 
motility  of,  35 
pathogenic  features  of,  100 
reproduction  of,  32 
size  of,  32 
staining  of,  42 
varieties  of,  29 

Bacterial    growth,    decomposable    or- 
ganic material,  36 
essential  conditions  of,  36 
heat,  36 
moisture,  39 
special  chemical  reaction  of  the 

culture-medium,  38 
life,  38 

inert  conditions  of,  38 
inhibitive  conditions  of,  38 
Bacterium,  28 
Bichloride  of  mercury  as  an  antiseptic, 

83 

Blood-serum  as  culture-media,  5(5,  72 
Blood  in  typhoid  fever,  preparing  spec- 
imen of.  163 

Boiling  water  as  an  antiseptic,  83 
Bouillon,  see  Culture-media. 
Bread-paste,  61 
Brownian  movements,  35 


INDEX. 


207 


Bubonic  plague,  176 
history  of,  176 

pANNON'S  influenza  bacillus,  174 
\J     Capsules,  staining  of,  47 
Johne's  method,  47 
Welch's  method,  47 
Carbolic  acid  as  an  antiseptic,  83 
Chamberlain's  filter,  81 
Chauveau's  retention  theory,  97 
Chemical  agents,  84 
Chemicals,  choice  of,  82 

use  of,  for  disinfecting,  81 
Chicken  cholera,  183 

cause  of,  183 
Chlorinated    lime    as    an    antiseptic, 

83 
Cholera  spirillum,  168 

artificial  susceptibility  to,  172 
diagnosis  of,  173 
diagnostic  test  of,  172 
growth  of,  171 
history  of,  168 
immunity  against,  172 
morphology  of,  168 
pathogeuesis  of,  171 
staining  of,  169 
vaccination  against,  173 
vitality  of,  171 
Clostridium,  33 
Coccus,  varieties  of,  29 
Cohn,  classification  of,  for  bacteria,  28 
Colonies,  counting  of,  71 
Comma   bacillus   (see    Cholera   spiril- 
lum), 168 
Control  test,  79 

Cultivation  of  anaerobic  bacteria,  72 
of  bacteria,  55 

utensils  used,  62,  63,  64,  65,  66,  67 
of  tetanus  bacillus,  73 
Culture-media,  agar,  59 
blood-serum,  56 
bouillon,  57 
bread-paste,  61 
Eisner's  medium,  160 
gelatin,  58 
glucose-bouillon,  62 
glycerin -agar,  60 
glycerin-bouillon,  60 
lactose-bouillon,  62 
milk,  55 

Pasteur's  solution,  57 
peptone  solution,  61 
potato,  60 
potato-paste,  61 


Culture-media,      saccharose-bouillon, 
62 

urine,  57 
Cultures,  agar  slant,  72 

blood-serum,  72 

diptheria  bacillus,  139 

from  secretions,  90 

human  body,  90 

thoracic  organs,  90 

DIMNESS,  tests  for  sources  of,  in  the 
object,  25 
Diphtheria,  133 
antitoxins,  142 

preparation  of,  142 
standardization  of,  142,  143 
treatment  of,  141  et  seq. 
bacillus,  133 

biologic  characters  of,  136 
cultures  of,  139,  140 
distribution  of,  134 
morphology  of,  134 
pathogen esis  of,  137,  138 
powers  of  resistance  of,  136 
staining  of,  134 

Neisser's  method,  135 
diagnosis  of,  138 
history  of,  133 
Diplococcus    intracellularis  meningi- 

tidis,  112 

biologic  characters  of,  113 
cultures  of,  from  man,  112 
discovery  of,  112 
morphology  of,  112 
pathogenesis  of,  113 
Disinfection,  methods  of,  81  et  seq. 
Drummer-bacillus,  33 
Dyes,  application  of,  43 

E BERT'S  bacillus,  156 
Ehrlich's  chain  theory,  98 
Eisner's  medium,  160,  161 
Examination   of  feces  in   dysentery, 

194 
of  water,  196  et  seq. 

FARCY,  124 
Fermentation,  38 

alcoholic  and  acetic  acid,  38 
butyric  and  lactic  acid,  38 
Fission,  32 
Flagella,  35 

staining  of,  49,  50 

Loefiler's  method,  49 
Formalin  as  an  antiseptic,  84 


208 


INDEX. 


GASES,  39 
Gelatin  media,  58 
Germicidal  power,  to  test,  82,  83 
Germicides  (see  Antiseptics),  81 
Glanders,  124 
Glycerin-agar  media,  60 
Gonococcus,  99,  104 
Gonorrhoaa,  104 
Gram's  method,  52 

HANGING- DROP,  42 
Heat,  sterilization  by,  79,  80 
Hog-cholera,  182 
Hyphomycetes  (mucorini),  28 

IMMUNITY,  94 
JL     acquired,  95 
active,  95 
methods  of,  95,  96 
natural,  94 
passive,  95 
theories  of,  97,  98 
Incubator,  75 
Infection,  associated,  94 
avenues  of,  92 
bacteria  in,  quantity  of,  93 
chemical  theory  of,  92 
definition  of,  91 
factors  of,  92  et  seq. 
mechanical  theory,  91 
Influenza,  174 
bacillus  of,  174 
biologic  characters  of,  175 
history  of,  174 
morphology  of,  174 
pathogen esis  of,  175 
vitality  of,  175 
Inoculation  of  animals,  85 
of  fluid  media,  68 
of  gelatin  culture  tubes,  69 
methods,  85  et  seq. 
of  solid  media,  68 

Intestinal  changes  in  dysentery,  181 
Intraperitoneal  inoculation,  88 
Intrapleural  inoculation,  88 
Intravenous  injection,  86,  87 

KITASATO  on  plague,  176 
Kitasato's  mouse-holder,  86 
Koch,  bacteriological  researches  of,  27 
Koch's   bacillus  of  rabbit  septicaemia, 

184 

sterilizer,  80 
tubercle  bacillus,  115 
tuberculin,  118 


Koch's  tuberculin  A,  O,  and  E,  119 
Kuehne's     carbolic     methylene-blue 
method,  53 

LAVERAN'S  malarial  parasite,  189 
Lens,  focus  of  a,  18 

type  of  objective,  23 
Lenses,  chromatic  aberration  of,  18 

objective,  21 

ocular,  21 

spherical  aberration  of,  18 
Leprosy,  120 

diagnosis  of,  122 

nature  of,  122 
Light,  direct,  20 

oblique,  20 

property  of  producing,  39 

refraction  of,  17 
Loeffler's  blood-serum,  56 

glanders  bacillus,  124 
Losch,  amoeba  of,  194 
Lustgarten's  bacillus,  122 
Lymphatics,  inoculation  into,  88 

MACROGAMETES,  191 
1V1     Malaria,  cycles  of,  189 
in  man,  189 
in  mosquito,  189 
Malarial  fever,  189 

examination  of  blood  in,  192 
mosquitoes  in,  189 
symptoms  of,  189 
parasite,  189 

characteristics  of,  190 
differentiation  of,  193 
inoculation  of,  193 
staining  of,  192 
varieties  of,  190 

Malignant  oedema,  bacillus  of,  152 
biologic  characters  of,  152 
morphology  of,  152 
pathogenesis  of,  152 
spores  of,  152 
Mallein,  127 

Mallory's  stain  (for  ray  fungus),  187 
Malta  fever,  113 
Melanin,  189 

MetchnikofTs  phagocytosis  theory,  97 
Methods,  special,  of  staining,  44 
Gabbett's,  46 
Gram's,  45 
Koch-Ehrlich's,  44 
Loeffler's,  44 
Ziehl's  carbol-fuchsin,  46 
Micrococcus  gonorrhoeas,  104 


INDEX. 


209 


Micrococcus  gonorrhoeas,  biologic  char- 
acters of,  105 
morphology  of,  104 
pathogenesis  of,  104 
staining  of,  105 
melitensis,  113 

biologic  characters  of,  113 
morphology  of,  113 
pathogenesis  of,  114 
pasteuri,  108 
pneumonias  crouposae,  108 

bioiogic  characters  of,  108 
history  of,  108 
immunization  of,  110 
intrathoracic  injection  of,  110 
morphology  of,  108 
subcutaneous  injection  of,  110 
pyogenes  tenuis  (Rosenbach),  99 
tetragenus,  99,  104 
morphology  of,  104 
pathogenesis  of,  104 
properties  of,  104 
Microgametocytes.  191 
Microscope,  care  of,  25 
compound,  19 
lenses  of,  17 
simple.  1!) 

working  distance  of,  23 
Milk  as  culture-medium,  r>.~> 

mode  of  preparing  sterilized,  50 
Monotrocha,  35 
Mordant,  50 

NATURAL  immunity,  94 
Neisser's  gonococcus,  104 
Nicolaier,  tetanus  bacillus  of,  145 
Numerical  aperture,  24 

ABERMEIER'S  spirillum,  179 

V     Objective,  angular  aperture  of  an, 

23 

to  cleanse,  25 
designation  of,  21 
Ocular,  to  cleanse  the  lenses  of,  25 
lens,  24 

types  of,  25 
(Edema,     malignant    (see    Malignant 

cedema),  152 
Optical  axis,  24 
Oxygen,  relation  of,  to  bacterial  life,  36 

PARASITES,  36 
Passet's    bacillus    pyogenes    foe- 

tidus.  10<> 
Passive  immunity,  95 

14— Bact. 


Pasteur,  bacteriological  researches  of, 

27 

Pasteur's  abstraction  theory,  97 
Pathogenic  bacteria,  99 
Pelvic  contents,  examination  of,  89 
Pepton  solution,  61 
Peritrocha.  35 
Pfeiffer's  bacillus,  174 
Phagocytosis  theory,  97 
Pitfield's  flagella  stain,  51 
Plasmodium  malariae,  189 
Plate  cultures  of  agar,  71 
Pneumobacillus  (Friedlaender's),  106 

p:ithogenesis,  107 
Pneumococcus,  99 

Friedlaender's,  110 

biologic  characters  of,  111 
discovery  of,  110 
morphology  of,  111 
pathogenesis  of,  111 
Pneumonia,  108 
Potato  as  culture-media,  60 

preparation  of,  for  test-tube  culture, 

61 
Pseudodiphtheria,  140 

differential  diagnosis  of,  141 
Ptomaines,  39 
Putrefaction,  causes  of,  39 

RAY  fungus,  186 
Refraction,  law  of,  17 
Reichert's  thermo-regulator,  75 
Relapsing  fever,  179 
Retention,  theory  of,  97 
Roux-Nocard  method  of  culture,  90 
history  of,  90 
importance  of,  90 
technic  of,  90 

QACCHAROMYCETES,       sprouting 

k3    fungi,  28 

Saprophytes,  36 

Schizomycetes,  cleft  fungi,  28 

Sedgwick-Tucker  method  for  examin- 
ing air,  203 

Septicaemia  sputum,  108 

Serotherapeutics,  178 

Scrum,  agglutinating  action  of,  178 
antityphoid,  165 

Shiga's  bacillus,  180 

Soil  examination  for  bacteria,  203 

Spirillum,  30 

cholera  Asiatics,  168 
Obermeieri,  179 
biology  of,  179 


210 


INDEX. 


Spirillum  Obermeieri,  history  of,  179 
morphology  of,  179 
pathogenesis  of,  180 
Spleen,  typhoid  bacilli  in,  161 
Spores,  staining  of,  47 

(Abbott)  first  method,  47 
second  method,  48    • 
third  method,  48 
(Fiocca)  fourth  method,  49 
Sporozoite,  191 
Sporulation,  32 

significance  of,  34 
Sputum  septicaemia,  108 
Staining,  33 
methods,  42,  116 

Bowhill's  method,  52 

Bunge's  method,  51 

Ehrlich's  modification  of  Koch's 

method,  44,  116 
Gabbett's  modification  of  Ziehl's 

method,  46,  116 
Koch's  method,  116 
Pitfield's  method,  51 
Van  Ermengem's  method,  52 
Ziehl's  carbol-fuchsin  method,  46, 

116 

Stains,  43 

Staphylococcus  cereus  albus,  99 
aureus  (Passet),  99 
flavus,  (Passet),  99 
pyogenes  albus,  101 
aureus,  100 
features  of,  100 
morphology  of,  100 
citreus,  102 

Sterilization,  definition  of,  76 
fractional,  77 
methods  of,  76  et  seq. 
Streptococcus  of  syphilis,  123 
Streptothrix,  185 
actinomyces  of,  186 
Eppingeri,  186  et  seq. 
madurse,  186 
pseudotuberculosa,  188 
resemblance  of,  to  bacteria,  185 

to  moulds,  185 
Subcutaneous  inoculation,  85 


Sulphur  dioxide  as  antiseptic,  83 
Syphilis,  122 
history  of,  122 

TETANIN,  148 
Tetanus,  145 
antitoxin,  151 
bacillus,  cultivation  of,  73 

history  of,  145 
toxin,  150 

Tissues,  staining  of  bacteria  in,  52 
Gram's  method,  52 
Kuehne's    carbolic   methylene- 

blue  method,  53 
Weigert's  method  (modification 

of  Gram),  53 

Ziehl-Neelsen's  method,  54 
Toxalbumins,  39 
Tuberculin  A,  O,  and  E,  119 

diagnosis  of  tuberculosis  by,  118 
Koch's,  118 
Tuberculosis,  115 
history  of,  115 
transmission  of,  118 
Typhoid  fever,  156 

vaccination  against,  164 

VAN  NEISSEN'S  streptococcus,  123 
Voges'  guinea-pig  holder,  86 

WATER,  bacillus  coli  communis  in, 
202 

bacteria  in,  196 

cholera  bacilli  in,  201 

counting  of  colonies  in,  198  et  seq. 

examination  of,  197 

pathogenic  germs  in,  201 

typhoid  bacilli  in,  201 
Weigert's     modification     of     Gram's 

method,   53 
Wiesnegg's  autoclave,  80 

EESIN  plague,  176 


7IEHL-NEELSEN'S  method,  54 

L 


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